Neprilysin inhibitors

ABSTRACT

In one aspect, the invention relates to compounds having the formula: 
                         
where R 1 , R 2 , R 3 , X, R 4 , R 5 , and R 6  are as defined in the specification, or a pharmaceutically acceptable salt thereof. These compounds have neprilysin inhibition activity. In another aspect, the invention relates to pharmaceutical compositions comprising such compounds; methods of using such compounds; and process and intermediates for preparing such compounds.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Application No. 61/491,749, filed on May 31, 2011; the entire disclosure of which is incorporated herein by reference.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to novel compounds having neprilysin-inhibition activity. The invention also relates to pharmaceutical compositions comprising such compounds, processes and intermediates for preparing such compounds and methods of using such compounds to treat diseases such as hypertension, heart failure, pulmonary hypertension, and renal disease.

2. State of the Art

Neprilysin (neutral endopeptidase, EC 3.4.24.11) (NEP), is an endothelial membrane bound Zn²⁺ metallopeptidase found in many organs and tissues, including the brain, kidneys, lungs, gastrointestinal tract, heart, and the peripheral vasculature. NEP degrades and inactivates a number of endogenous peptides, such as enkephalins, circulating bradykinin, angiotensin peptides, and natriuretic peptides, the latter of which have several effects including, for example, vasodilation and natriuresis/diuresis, as well as inhibition of cardiac hypertrophy and ventricular fibrosis. Thus, NEP plays an important role in blood pressure homeostasis and cardiovascular health.

NEP inhibitors, such as thiorphan, candoxatril, and candoxatrilat, have been studied as potential therapeutics. Compounds that inhibit both NEP and angiotensin-I converting enzyme (ACE) are also known, and include omapatrilat, gempatrilat, and sampatrilat. Referred to as vasopeptidase inhibitors, this latter class of compounds is described in Robl et al. (1999) Exp. Opin. Ther. Patents 9(12): 1665-1677.

SUMMARY OF THE INVENTION

The present invention provides novel compounds that have been found to possess neprilysin (NEP) enzyme inhibition activity. Accordingly, compounds of the invention are expected to be useful and advantageous as therapeutic agents for treating conditions such as hypertension and heart failure.

One aspect of the invention relates to a compound of formula I:

where:

R¹ is —OR¹⁰ or —NR⁶⁰R⁷⁰;

R² is H or —OR²⁰;

R³ is selected from H, Cl, F, —CH₃, and —CF₃;

X is a —C₁₋₁₀heteroaryl or a partially unsaturated —C₂₋₁₂heterocycle, with the proviso that X is not pyrazole;

R⁴ is absent or is selected from H; halo; —C₀₋₅alkylene-OH; —NH₂; —C₁₋₆alkyl; —CF₃; —C₃₋₇cycloalkyl; —C₀₋₂alkylene-O—C₁₋₆alkyl; —C(O)R⁴⁰; —C₀₋₁alkylene-C(O)OR⁴¹; —C(O)NR⁴²R⁴³; —NHC(O)R⁴⁴; ═O; —NO₂; furan; pyrazine; naphthalene; pyridine; pyrazole optionally substituted with methyl; thiophene optionally substituted with methyl; and phenyl optionally substituted with one group selected from halo, —OH, —CF₃, —OCH₃, —NHC(O)CH₃, and phenyl; and R⁴, when present, is attached to a carbon atom; and R⁴ can also be phenyl substituted with —C(O)OR⁴¹ when X is pyridazine, pyrazine, or pyridine;

R⁵ is absent or is selected from H; —C₁₋₆alkyl; —C₀₋₃alkylene-OH; —[(CH₂)₂O]₁₋₃CH₃; —C₁₋₃alkylene-C(O)OR⁵⁰; —CH₂—C(O)NR⁵¹R⁵²; —C₀₋₂alkylene-pyridine optionally substituted with halo; —CH₂-isoxazole optionally substituted with methyl; —CH₂-pyrimidine optionally substituted with —O—C₁₋₆alkyl; —C₂alkylene-phenyl;

and R⁵, when present, is attached to a nitrogen atom;

R⁶ is absent or is selected from H, halo, —OH, —C₁₋₆alkyl, and —O—C₁₋₆alkyl; and R⁶, when present, is attached to a carbon atom;

R¹⁰, R⁴¹ and R⁵⁰ are independently selected from H, —C₁₋₆alkyl, —C₁₋₃alkylene-C₆₋₁₀aryl, —C₁₋₃alkylene-C₁₋₉heteroaryl, —C₃₋₇cycloalkyl, —[(CH₂)₂O]₁₋₃CH₃, —C₁₋₆alkylene-OC(O)R¹³, —C₁₋₆alkylene-NR¹⁴R¹⁵, —C₁₋₆alkylene-C(O)R¹⁷, —C₀₋₆alkylenemorpholine, —C₁₋₆alkylene-SO₂—C₁₋₆alkyl,

R¹³ is selected from —C₁₋₆alkyl, —O—C₁₋₆alkyl, —C₃₋₇cycloalkyl, —O—C₃₋₇cycloalkyl, phenyl, —O-phenyl, —NR¹⁴R¹⁵, and —CH(NH₂)CH₂COOCH₃; R¹⁴ and R¹⁵ are independently selected from H, —C₁₋₆alkyl, and benzyl, or R¹⁴ and R¹⁵ are taken together as —(CH₂)₃₋₆—, —C(O)—(CH₂)₃—, or —(CH₂)₂O(CH₂)₂—; R¹⁶ is —C₁₋₆alkyl or —C₀₋₆alkylene-C₆₋₁₀aryl; R¹⁷ is selected from —O—C₁₋₆alkyl, —O-benzyl, and —NR¹⁴R¹⁵;

R²⁰ is H or is taken together with R¹⁰ to form —CR²¹R²²— or is taken together with R⁶⁰ to form —C(O)—; where R²¹ and R²² are independently selected from H, —C₁₋₆alkyl, and —O—C₃₋₇cycloalkyl, or R²¹ and R²² are taken together to form ═O;

R⁴⁰ is H or —C₁₋₆alkyl;

R⁴², R⁴³, R⁵¹, and R⁵² are independently selected from H, —C₁₋₆alkyl, —CH₂COOH, —(CH₂)₂OH, —(CH₂)₂OCH₃, —(CH₂)₂SO₂NH₂, —(CH₂)₂N(CH₃)₂, —C₃₋₇cycloalkyl, and —(CH₂)₂-imidazole; or R⁴² and R⁴³ or R⁵² and R⁵³ are taken together to form a saturated or partially unsaturated —C₃₋₅heterocycle optionally substituted with —OH, —COOR⁴¹, or —CONH₂, and optionally containing an oxygen atom in the ring;

R⁴⁴ is selected from —C₁₋₆alkyl; —C₀₋₁alkylene-O—C₁₋₆alkyl; phenyl optionally substituted with halo or —OCH₃; and —C₁₋₉heteroaryl; and

R⁵³, R⁵⁴, R⁵⁵, R⁵⁶, and R⁵⁷ are independently selected from H, halo, —C₁₋₆alkyl, —O—C₁₋₆alkyl, and —S—C₁₋₆alkyl, where each —C₁₋₆alkyl is optionally substituted with 1-5 fluoro atoms;

R⁶⁰ is selected from H, —OH, —OC(O)R⁶¹, —CH₂COOH, —O-benzyl, pyridyl, and —OC(S)NR⁶²R⁶³; where R⁶¹ is selected from H, —C₁₋₆alkyl, —C₆₋₁₀aryl, —OCH₂—C₆₋₁₀aryl, —CH₂O—C₆₋₁₀aryl, and —NR⁶²R⁶³; R⁶² and R⁶³ are independently H or —C₁₋₄alkyl;

R⁷⁰ is selected from H, —C₁₋₆alkyl, and —C(O)R⁷¹; R⁷¹ is selected from H, —C₁₋₆alkyl, —C₃₋₇cycloalkyl, —C₆₋₁₀aryl, and —C₁₋₉heteroaryl;

or a pharmaceutically acceptable salt thereof.

Another aspect of the invention relates to pharmaceutical compositions comprising a pharmaceutically acceptable carrier and a compound of the invention. Such compositions may optionally contain other therapeutic agents. Accordingly, in yet another aspect of the invention, a pharmaceutical composition comprises a compound of the invention as the first therapeutic agent, one or more secondary therapeutic agent, and a pharmaceutically acceptable carrier. Another aspect of the invention relates to a combination of active agents, comprising a compound of the invention and a second therapeutic agent. The compound of the invention can be formulated together or separately from the additional agent(s). When formulated separately, a pharmaceutically acceptable carrier may be included with the additional agent(s). Thus, yet another aspect of the invention relates to a combination of pharmaceutical compositions, the combination comprising: a first pharmaceutical composition comprising a compound of the invention and a first pharmaceutically acceptable carrier; and a second pharmaceutical composition comprising a second therapeutic agent and a second pharmaceutically acceptable carrier. In another aspect, the invention relates to a kit containing such pharmaceutical compositions, for example where the first and second pharmaceutical compositions are separate pharmaceutical compositions.

Compounds of the invention possess NEP enzyme inhibition activity, and are therefore expected to be useful as therapeutic agents for treating patients suffering from a disease or disorder that is treated by inhibiting the NEP enzyme or by increasing the levels of its peptide substrates. Thus, one aspect of the invention relates to a method of treating patients suffering from a disease or disorder that is treated by inhibiting the NEP enzyme, comprising administering to a patient a therapeutically effective amount of a compound of the invention. Another aspect of the invention relates to a method of treating hypertension, heart failure, or renal disease, comprising administering to a patient a therapeutically effective amount of a compound of the invention. Still another aspect of the invention relates to a method for inhibiting a NEP enzyme in a mammal comprising administering to the mammal, a NEP enzyme-inhibiting amount of a compound of the invention.

Since compounds of the invention possess NEP inhibition activity, they are also useful as research tools. Accordingly, one aspect of the invention relates to a method of using a compound of the invention as a research tool, the method comprising conducting a biological assay using a compound of the invention. Compounds of the invention can also be used to evaluate new chemical compounds. Thus another aspect of the invention relates to a method of evaluating a test compound in a biological assay, comprising: (a) conducting a biological assay with a test compound to provide a first assay value; (b) conducting the biological assay with a compound of the invention to provide a second assay value; wherein step (a) is conducted either before, after or concurrently with step (b); and (c) comparing the first assay value from step (a) with the second assay value from step (b). Exemplary biological assays include a NEP enzyme inhibition assay. Still another aspect of the invention relates to a method of studying a biological system or sample comprising a NEP enzyme, the method comprising: (a) contacting the biological system or sample with a compound of the invention; and (b) determining the effects caused by the compound on the biological system or sample.

Yet another aspect of the invention relates to processes and intermediates useful for preparing compounds of the invention. Accordingly, another aspect of the invention relates to a process of preparing compounds of formula I, comprising the steps of: (a) coupling compound 1 with compound 2:

(b) coupling compound 1 with compound 2a to form compound 3:

where L is a leaving group such as a halogen (e.g., bromo) or a triflate (e.g., —OSO₂CF₃), and reacting compound 3 with optionally substituted 4,4,5,5-tetramethyl-2-phenyl-[1,3,2]dioxaborolane:

in a palladium-catalyzed coupling reaction; or (c) reacting compound 2b with optionally substituted 4,4,5,5-tetramethyl-2-phenyl-[1,3,2]dioxaborolane to form compound 4 in a palladium-catalyzed coupling reaction:

where L is a leaving group and P¹ is a carboxy-protecting group selected from methyl, ethyl, t-butyl, benzyl, p-methoxybenzyl, 9-fluorenylmethyl, trimethylsilyl, t-butyldimethylsilyl, and diphenylmethyl, and coupling compound 4 with compound 1; and (d) optionally deprotecting the product of step (a) or (b) or (c), to produce a compound of formula I or a pharmaceutically acceptable salt thereof; where R¹, R², R³, X, R⁴, R⁵, and R⁶ are as defined for formula I. Another aspect of the invention relates to a process of preparing a pharmaceutically acceptable salt of a compound of formula I, comprising contacting a compound of formula I in free acid or base form with a pharmaceutically acceptable base or acid. In other aspects, the invention relates to products prepared by any of the processes described herein, as well as novel intermediates used in such process. In one aspect of the invention novel intermediates have formula II, as defined herein.

Yet another aspect of the invention relates to the use of a compound of formula I or a pharmaceutically acceptable salt thereof, for the manufacture of a medicament, especially for the manufacture of a medicament useful for treating hypertension, heart failure, or renal disease. Another aspect of the invention relates to use of a compound of the invention for inhibiting a NEP enzyme in a mammal Still another aspect of the invention relates to the use of a compound of the invention as a research tool. Other aspects and embodiments of the invention are disclosed herein.

DETAILED DESCRIPTION OF THE INVENTION Definitions

When describing the compounds, compositions, methods and processes of the invention, the following terms have the following meanings unless otherwise indicated. Additionally, as used herein, the singular forms “a,” “an,” and “the” include the corresponding plural forms unless the context of use clearly dictates otherwise. The terms “comprising”, “including,” and “having” are intended to be inclusive and mean that there may be additional elements other than the listed elements. All numbers expressing quantities of ingredients, properties such as molecular weight, reaction conditions, and so forth used herein are to be understood as being modified in all instances by the term “about,” unless otherwise indicated. Accordingly, the numbers set forth herein are approximations that may vary depending upon the desired properties sought to be obtained by the present invention. At least, and not as an attempt to limit the application of the doctrine of equivalents to the scope of the claims, each number should at least be construed in light of the reported significant digits and by applying ordinary rounding techniques.

The term “alkyl” means a monovalent saturated hydrocarbon group which may be linear or branched. Unless otherwise defined, such alkyl groups typically contain from 1 to 10 carbon atoms and include, for example, —C₁₋₄alkyl, —C₁₋₅alkyl, —C₂₋₅alkyl, —C₁₋₆alkyl, and —C₁₋₁₀alkyl. Representative alkyl groups include, by way of example, methyl, ethyl, n-propyl, isopropyl, n-butyl, s-butyl, isobutyl, t-butyl, n-pentyl, n-hexyl, n-heptyl, n-octyl, n-nonyl, n-decyl, and the like.

When a specific number of carbon atoms is intended for a particular term used herein, the number of carbon atoms is shown preceding the term as subscript. For example, the term “—C₁₋₆alkyl” means an alkyl group having from 1 to 6 carbon atoms, and the term “—C₃₋₇cycloalkyl” means a cycloalkyl group having from 3 to 7 carbon atoms, respectively, where the carbon atoms are in any acceptable configuration.

The term “alkylene” means a divalent saturated hydrocarbon group that may be linear or branched. Unless otherwise defined, such alkylene groups typically contain from 0 to 10 carbon atoms and include, for example, —C₀₋₁alkylene-, —C₀₋₆alkylene-, and —C₁₋₃alkylene-. Representative alkylene groups include, by way of example, methylene, ethane-1,2-diyl (“ethylene”), propane-1,2-diyl, propane-1,3-diyl, butane-1,4-diyl, pentane-1,5-diyl and the like. It is understood that when the alkylene term include zero carbons such as —C₀₋₁alkylene-, such terms are intended to include the absence of carbon atoms, that is, the alkylene group is not present except for a covalent bond attaching the groups separated by the alkylene term.

The term “aryl” means a monovalent aromatic hydrocarbon having a single ring (i.e., phenyl) or one or more fused rings. Fused ring systems include those that are fully unsaturated (e.g., naphthalene) as well as those that are partially unsaturated (e.g., 1,2,3,4-tetrahydronaphthalene). Unless otherwise defined, such aryl groups typically contain from 6 to 10 carbon ring atoms and include, for example, —C₆₋₁₀aryl. Representative aryl groups include, by way of example, phenyl and naphthalene-1-yl, naphthalene-2-yl, and the like.

The term “cycloalkyl” means a monovalent saturated carbocyclic hydrocarbon group. Unless otherwise defined, such cycloalkyl groups typically contain from 3 to 10 carbon atoms and include, for example, —C₃₋₅cycloalkyl, —C₃₋₆cycloalkyl and —C₃₋₇cycloalkyl. Representative cycloalkyl groups include, by way of example, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl and the like.

The term “halo” means fluoro, chloro, bromo and iodo.

The term “heterocycle” is intended to include monovalent unsaturated (aromatic) heterocycles having a single ring or two fused rings as well as monovalent saturated and partially unsaturated groups having a single ring or multiple condensed rings. The heterocycle ring can contain from 3 to 15 total ring atoms, of which 1 to 14 are ring carbon atoms, and 1 to 4 are ring heteroatoms selected from nitrogen, oxygen or sulfur. Typically, however, the heterocycle ring contains from 3 to 10 total ring atoms, of which 1 to 9 are ring carbon atoms, and 1 to 4 are ring heteroatoms. The point of attachment is at any available carbon or nitrogen ring atom. Exemplary heterocycles include, for example, —C₁₋₇heterocycle, —C₃₋₅heterocycle, —C₂₋₆heterocycle, —C₃₋₁₂heterocycle, —C₅₋₉heterocycle, —C₁₋₉heterocycle, —C₁₋₁₁heterocycle, and —C₁₋₁₄heterocyle.

Monovalent unsaturated heterocycles are also commonly referred to as “heteroaryl” groups. Unless otherwise defined, heteroaryl groups typically contain from 5 to 10 total ring atoms, of which 1 to 9 are ring carbon atoms, and 1 to 4 are ring heteroatoms, and include, for example, —C₁₋₉heteroaryl and —C₅₋₉heteroaryl. Representative heteroaryl groups include, by way of example, pyrrole (e.g., 3-pyrrolyl and 2H-pyrrol-3-yl), imidazole (e.g., 2-imidazolyl), furan (e.g., 2-furyl and 3-furyl), thiophene (e.g., 2-thienyl), triazole (e.g., 1,2,3-triazolyl and 1,2,4-triazolyl), pyrazole (e.g., 1H-pyrazol-3-yl), oxazole (e.g., 2-oxazolyl), isoxazole (e.g., 3-isoxazolyl), thiazole (e.g., 2-thiazolyl and 4-thiazolyl), and isothiazole (e.g., 3-isothiazolyl), pyridine (e.g., 2-pyridyl, 3-pyridyl, and 4-pyridyl), pyridylimidazole, pyridyltriazole, pyrazine, pyridazine (e.g., 3-pyridazinyl), pyrimidine (e.g., 2-pyrimidinyl), tetrazole, triazine (e.g., 1,3,5-triazinyl), indolyle (e.g., 1H-indol-2-yl, 1H-indol-4-yl and 1H-indol-5-yl), benzofuran (e.g., benzofuran-5-yl), benzothiophene (e.g., benzo[b]thien-2-yl and benzo[b]thien-5-yl), benzimidazole, benzoxazole, benzothiazole, benzotriazole, quinoline (e.g., 2-quinolyl), isoquinoline, quinazoline, quinoxaline and the like.

Monovalent saturated heterocycles typically contain from 3 to 10 total ring atoms, of which 2 to 9 are ring carbon atoms, and 1 to 4 are ring heteroatoms, and include, for example —C₃₋₅heterocycle. Representative monovalent saturated heterocycles include, by way of example, monovalent species of pyrrolidine, imidazolidine, pyrazolidine, piperidine, 1,4-dioxane, morpholine, thiomorpholine, piperazine, 3-pyrroline and the like. In some instances, moieties may be described as being taken together to form a saturated —C₃₋₅heterocycle optionally containing an oxygen atom in the ring. Such groups include:

Monovalent partially unsaturated heterocycles typically contain from 3 to 10 total ring atoms, of which 2 to 11 are ring carbon atoms, and 1 to 3 are ring heteroatoms, and include, for example —C₃₋₅heterocycle and —C₂₋₁₂heterocycle. Representative monovalent partially unsaturated heterocycles include, by way of example, pyran, benzopyran, benzodioxole (e.g., benzo[1,3]dioxol-5-yl), tetrahydropyridazine, 2,5-dihydro-1H-pyrrole, dihydroimidazole, dihydrotriazole, dihydrooxazole, dihydroisoxazole, dihydrothiazole, dihydroisothiazole, dihydrooxadiazole, dihydrothiadiazole, tetrahydropyridazine, hexahydropyrroloquinoxaline, and dihydrooxadiazabenzo[e]azulene. In some instances, moieties may be described as being taken together to form a partially unsaturated —C₃₋₅heterocycle. Such groups include:

The term “optionally substituted” means that group in question may be unsubstituted or it may be substituted one or several times, such as 1 to 3 times or 1 to 5 times. For example, a phenyl group that is “optionally substituted” with halo atoms, may be unsubstituted, or it may contain 1, 2, 3, 4, or 5 halo atoms.

As used herein, the phrase “having the formula” or “having the structure” is not intended to be limiting and is used in the same way that the term “comprising” is commonly used.

The term “pharmaceutically acceptable” refers to a material that is not biologically or otherwise unacceptable when used in the invention. For example, the term “pharmaceutically acceptable carrier” refers to a material that can be incorporated into a composition and administered to a patient without causing unacceptable biological effects or interacting in an unacceptable manner with other components of the composition. Such pharmaceutically acceptable materials typically have met the required standards of toxicological and manufacturing testing, and include those materials identified as suitable inactive ingredients by the U.S. Food and Drug administration.

The term “pharmaceutically acceptable salt” means a salt prepared from a base or an acid which is acceptable for administration to a patient, such as a mammal (for example, salts having acceptable mammalian safety for a given dosage regime). However, it is understood that the salts covered by the invention are not required to be pharmaceutically acceptable salts, such as salts of intermediate compounds that are not intended for administration to a patient. Pharmaceutically acceptable salts can be derived from pharmaceutically acceptable inorganic or organic bases and from pharmaceutically acceptable inorganic or organic acids. In addition, when a compound of formula I contains both a basic moiety, such as an amine, pyridine or imidazole, and an acidic moiety such as a carboxylic acid or tetrazole, zwitterions may be formed and are included within the term “salt” as used herein. Salts derived from pharmaceutically acceptable inorganic bases include ammonium, calcium, copper, ferric, ferrous, lithium, magnesium, manganic, manganous, potassium, sodium, and zinc salts, and the like. Salts derived from pharmaceutically acceptable organic bases include salts of primary, secondary and tertiary amines, including substituted amines, cyclic amines, naturally-occurring amines and the like, such as arginine, betaine, caffeine, choline, N,N′-dibenzylethylenediamine, diethylamine, 2-diethylaminoethanol, 2-dimethylaminoethanol, ethanolamine, ethylenediamine, N-ethylmorpholine, N-ethylpiperidine, glucamine, glucosamine, histidine, hydrabamine, isopropylamine, lysine, methylglucamine, morpholine, piperazine, piperadine, polyamine resins, procaine, purines, theobromine, triethylamine, trimethylamine, tripropylamine, tromethamine and the like. Salts derived from pharmaceutically acceptable inorganic acids include salts of boric, carbonic, hydrohalic (hydrobromic, hydrochloric, hydrofluoric or hydroiodic), nitric, phosphoric, sulfamic and sulfuric acids. Salts derived from pharmaceutically acceptable organic acids include salts of aliphatic hydroxyl acids (for example, citric, gluconic, glycolic, lactic, lactobionic, malic, and tartaric acids), aliphatic monocarboxylic acids (for example, acetic, butyric, formic, propionic and trifluoroacetic acids), amino acids (for example, aspartic and glutamic acids), aromatic carboxylic acids (for example, benzoic, p-chlorobenzoic, diphenylacetic, gentisic, hippuric, and triphenylacetic acids), aromatic hydroxyl acids (for example, o-hydroxybenzoic, p-hydroxybenzoic, 1-hydroxynaphthalene-2-carboxylic and 3-hydroxynaphthalene-2-carboxylic acids), ascorbic, dicarboxylic acids (for example, fumaric, maleic, oxalic and succinic acids), glucoronic, mandelic, mucic, nicotinic, orotic, pamoic, pantothenic, sulfonic acids (for example, benzenesulfonic, camphosulfonic, edisylic, ethanesulfonic, isethionic, methanesulfonic, naphthalenesulfonic, naphthalene-1,5-disulfonic, naphthalene-2,6-disulfonic and p-toluenesulfonic acids), xinafoic acid, and the like.

As used herein, the term “prodrug” is intended to mean an inactive (or significantly less active) precursor of a drug that is converted into its active form in the body under physiological conditions, for example, by normal metabolic processes. The term is also intended to include certain protected derivatives of compounds of formula I that may be made prior to a final deprotection stage. Such compounds may not possess pharmacological activity at NEP, but may be administered orally or parenterally and thereafter metabolized in the body to form compounds of the invention which are pharmacologically active at NEP. Thus, all protected derivatives and prodrugs of compounds formula I are included within the scope of the invention. Prodrugs of compounds of formula I having a free carboxyl group can be readily synthesized by techniques that are well known in the art. These prodrug derivatives are then converted by solvolysis or under physiological conditions to be the free carboxyl compound. Exemplary prodrugs include esters such as C₁₋₆alkylesters and aryl-C₁₋₆alkylesters. In one embodiment, the compounds of formula I have a free carboxyl and the prodrug is an ester derivative thereof, i.e., the prodrug is an ester such as —C(O)OCH₂CH₃.

The term “therapeutically effective amount” means an amount sufficient to effect treatment when administered to a patient in need thereof, that is, the amount of drug needed to obtain the desired therapeutic effect. For example, a therapeutically effective amount for treating hypertension is an amount of compound needed to, for example, reduce, suppress, eliminate, or prevent the symptoms of hypertension, or to treat the underlying cause of hypertension. In one embodiment, a therapeutically effective amount is that amount of drug needed to reduce blood pressure or the amount of drug needed to maintain normal blood pressure. On the other hand, the term “effective amount” means an amount sufficient to obtain a desired result, which may not necessarily be a therapeutic result. For example, when studying a system comprising a NEP enzyme, an “effective amount” may be the amount needed to inhibit the enzyme.

The term “treating” or “treatment” as used herein means the treating or treatment of a disease or medical condition (such as hypertension) in a patient, such as a mammal (particularly a human) that includes one or more of the following: (a) preventing the disease or medical condition from occurring, i.e., preventing the reoccurrence of the disease or medical condition or prophylactic treatment of a patient that is pre-disposed to the disease or medical condition; (b) ameliorating the disease or medical condition, i.e., eliminating or causing regression of the disease or medical condition in a patient; (c) suppressing the disease or medical condition, i.e., slowing or arresting the development of the disease or medical condition in a patient; or (d) alleviating the symptoms of the disease or medical condition in a patient. For example, the term “treating hypertension” would include preventing hypertension from occurring, ameliorating hypertension, suppressing hypertension, and alleviating the symptoms of hypertension (for example, lowering blood pressure). The term “patient” is intended to include those mammals, such as humans, that are in need of treatment or disease prevention or that are presently being treated for disease prevention or treatment of a specific disease or medical condition, as well as test subjects in which compounds of the invention are being evaluated or being used in an assay, for example an animal model.

All other terms used herein are intended to have their ordinary meaning as understood by those of ordinary skill in the art to which they pertain.

In one aspect, the invention relates to compounds of formula I:

or a pharmaceutically acceptable salt thereof.

As used herein, the term “compound of the invention” includes all compounds encompassed by formula I such as the species embodied in formulas Ia-Il, as well as the compounds encompassed by formula II. In addition, the compounds of the invention may also contain several basic or acidic groups (for example, amino or carboxyl groups) and therefore, such compounds can exist as a free base, free acid, or in various salt forms. All such salt forms are included within the scope of the invention. Furthermore, the compounds of the invention may also exist as prodrugs. Accordingly, those skilled in the art will recognize that reference to a compound herein, for example, reference to a “compound of the invention” or a “compound of formula I” includes a compound of formula I as well as pharmaceutically acceptable salts and prodrugs of that compound unless otherwise indicated. Further, the term “or a pharmaceutically acceptable salt and/or prodrug thereof” is intended to include all permutations of salts and prodrugs, such as a pharmaceutically acceptable salt of a prodrug. Furthermore, solvates of compounds of formula I are included within the scope of this invention.

The compounds of formula I may contain one or more chiral centers and therefore, these compounds may be prepared and used in various stereoisomeric forms. Accordingly, the invention also relates to racemic mixtures, pure stereoisomers (e.g., enantiomers and diastereoisomers), stereoisomer-enriched mixtures, and the like unless otherwise indicated. When a chemical structure is depicted herein without any stereochemistry, it is understood that all possible stereoisomers are encompassed by such structure. Thus, for example, the terms “compound of formula I,” “compounds of formula II,” and so forth, are intended to include all possible stereoisomers of the compound. Similarly, when a particular stereoisomer is shown or named herein, it will be understood by those skilled in the art that minor amounts of other stereoisomers may be present in the compositions of the invention unless otherwise indicated, provided that the utility of the composition as a whole is not eliminated by the presence of such other isomers. Individual stereoisomers may be obtained by numerous methods that are well known in the art, including chiral chromatography using a suitable chiral stationary phase or support, or by chemically converting them into diastereoisomers, separating the diastereoisomers by conventional means such as chromatography or recrystallization, then regenerating the original stereoisomer.

Additionally, where applicable, all cis-trans or E/Z isomers (geometric isomers), tautomeric forms and topoisomeric forms of the compounds of the invention are included within the scope of the invention unless otherwise specified. For example, if X is depicted as (R⁵ being hydrogen):

it is understood that the compound may also exist in a tautomeric form such as:

More specifically, compounds of formula I contain at least one chiral center when R² is H, and contain at least two chiral centers when R² is —OR²⁰. These chiral centers are indicated by the symbols * and ** in the following formulas Ia and Ib:

In one stereoisomer of the compounds of formula Ia, the carbon atom identified by the ** symbol has the (R) configuration. This embodiment of the invention is shown in formula Ia-1:

In this embodiment, compounds have the (R) configuration at the ** carbon atom or are enriched in a stereoisomeric form having the (R) configuration at this carbon atom. In another stereoisomer of the compounds of formula Ia, the carbon atom identified by the ** symbol has the (S) configuration. This embodiments of the invention is shown in formula Ia-2:

In this embodiment, compounds have the (S) configuration at the ** carbon atom or are enriched in a stereoisomeric form having the (S) configuration at this carbon atom.

In one stereoisomer of the compound of formula Ib, both carbon atoms identified by the * and ** symbols have the (R) configuration. This embodiment of the invention is shown in formula Ib-1:

In this embodiment, compounds have the (R,R) configuration at the * and ** carbon atoms or are enriched in a stereoisomeric form having the (R,R) configuration at these carbon atoms. In another stereoisomer of the compound of formula Ib, both carbon atoms identified by the * and ** symbols have the (S) configuration. This embodiment of the invention is shown in formula Ib-2:

In this embodiment, compounds have the (S,S) configuration at the * and ** carbon atoms or are enriched in a stereoisomeric form having the (S,S) configuration at these carbon atoms. In yet another stereoisomer of the compound of formula Ib, the carbon atom identified by the symbol * has the (S) configuration and the carbon atom identified by the symbol ** has the (R) configuration. This embodiment of the invention is shown in formula Ib-3:

In this embodiment, compounds have the (S,R) configuration at the * and ** carbon atoms or are enriched in a stereoisomeric form having the (S,R) configuration at these carbon atoms. In still another stereoisomer of the compound of formula Ib, the carbon atom identified by the symbol * has the (R) configuration and the carbon atom identified by the symbol ** has the (S) configuration. This embodiment of the invention is shown in formula Ib-4:

In this embodiment, compounds have the (R,S) configuration at the * and ** carbon atoms or are enriched in a stereoisomeric form having the (R,S) configuration at these carbon atoms.

The compounds of the invention, as well as those compounds used in their synthesis, may also include isotopically-labeled compounds, that is, where one or more atoms have been enriched with atoms having an atomic mass different from the atomic mass predominately found in nature. Examples of isotopes that may be incorporated into the compounds of formula I, for example, include, but are not limited to, ²H, ³H, ¹³C, ¹⁴C, ¹⁵N, ¹⁸O, ¹⁷O, ³⁵S, ³⁶Cl, and ¹⁸F. Of particular interest are compounds of formula I enriched in tritium or carbon-14 which can be used, for example, in tissue distribution studies; compounds of formula I enriched in deuterium especially at a site of metabolism resulting, for example, in compounds having greater metabolic stability; and compounds of formula I enriched in a positron emitting isotope, such as ¹¹C, ¹⁸F, ¹⁵O and ¹³N, which can be used, for example, in Positron Emission Topography (PET) studies.

The nomenclature used herein to name the compounds of the invention is illustrated in the Examples herein. This nomenclature has been derived using the commercially available AutoNom software (MDL, San Leandro, Calif.).

Representative Embodiments

The following substituents and values are intended to provide representative examples of various aspects and embodiments of the invention. These representative values are intended to further define and illustrate such aspects and embodiments and are not intended to exclude other embodiments or to limit the scope of the invention. In this regard, the representation that a particular value or substituent is preferred is not intended in any way to exclude other values or substituents from the invention unless specifically indicated.

In one embodiment, this invention relates to compounds of formula I:

R¹ is selected from —OR¹⁰ and —NR⁶⁰R⁷⁰. The R¹⁰ moiety is selected from H, —C₁₋₆alkyl, —C₁₋₃alkylene-C₆₋₁₀aryl, —C₁₋₃alkylene-C₁₋₉heteroaryl, —C₃₋₇cycloalkyl, —[(CH₂)₂O]₁₋₃CH₃, —C₁₋₆alkylene-OC(O)R¹³, —C₁₋₆alkylene-NR¹⁴R¹⁵, —C₁₋₆alkylene-C(O)R¹⁷, —C₀₋₆alkylenemorpholine, —C₁₋₆alkylene-SO₂—C₁₋₆alkyl,

where R¹³ is selected from —C₁₋₆alkyl, —O—C₁₋₆alkyl, —C₃₋₇cycloalkyl, —O—C₃₋₇cycloalkyl, phenyl, —O-phenyl, —NR¹⁴R¹⁵, and —CH(NH₂)CH₂COOCH₃; R¹⁴ and R¹⁵ are independently selected from H, —C₁₋₆alkyl, and benzyl, or R¹⁴ and R¹⁵ are taken together as —(CH₂)₃₋₆—, —C(O)—(CH₂)₃—, or —(CH₂)₂—O(CH₂)₂—; R¹⁶ is —C₁₋₆alkyl or —C₀₋₆alkylene-C₆₋₁₀aryl; and R¹⁷ is selected from —O—C₁₋₆alkyl, —O-benzyl, and —NR¹⁴R¹⁵. The R⁶⁰ moiety is selected from H, —OH, —OC(O)R⁶¹, —CH₂COOH, —O-benzyl, pyridyl, and —OC(S)NR⁶²R⁶³; where R⁶¹ is selected from H, —C₁₋₆alkyl, —C₆₋₁₀aryl, —OCH₂—C₆₋₁₀aryl, —CH₂O—C₆₋₁₀aryl, and —NR⁶²R⁶³; and R⁶² and R⁶³ are independently H or —C₁₋₄alkyl. The R⁷⁰ moiety is selected from H, —C₁₋₆alkyl, and —C(O)R⁷¹; where R⁷¹ is selected from H, —C₁₋₆alkyl, —C₃₋₇cycloalkyl, —C₆₋₁₀aryl, and —C₁₋₉heteroaryl.

In one embodiment, R¹ is —OR¹⁰, and R¹⁰ is H or —C₁₋₆alkyl (e.g., —CH₂CH₃ and —(CH₂)₃CH₃).

In one embodiment, R¹ is selected from —OR¹⁰ and —NR⁶⁰R⁷⁰, where R¹⁰ is H, R⁶⁰ is H or —OH, and R⁷⁰ is H.

In another embodiment, R¹ is —OR¹⁰, where R¹⁰ is selected from —C₁₋₆alkyl (e.g., —CH₂CH₃, —(CH₂)₂CH₃, —CH(CH₃)₂, —CH₂CH(CH₃)₂, —(CH₂)₃CH₃, —(CH₂)₄CH₃, and —(CH₂)₂CH(CH₃)₂), —C₁₋₃alkylene-C₆₋₁₀aryl (e.g., benzyl), —C₁₋₃alkylene-C₁₋₉heteroaryl, —C₃₋₇cycloalkyl, —[(CH₂)₂O]₁₋₃CH₃ (e.g., —(CH₂)₂OCH₃ and —[(CH₂)₂O]₂CH₃), —C₁₋₆alkylene- OC(O)R¹³ (e.g., —CH₂—OC(O)CH₃, —CH₂—OC(O)CH₂CH₃, and —CH₂—OC(O)OCH₂CH₃), —C₁₋₆alkylene-NR¹⁴R¹⁵ (e.g., —(CH₂)₂—N(CH₃)₂,

—C₁₋₆alkylene-C(O)R¹⁷ (e.g., —CH₂C(O)OCH₃, —CH₂C(O)O-benzyl, —CH₂C(O)—N(CH₃)₂, and

—C₀₋₆alkylenemorpholine, —C₁₋₆alkylene-SO₂—C₁₋₆alkyl (e.g., —(CH₂)₂SO₂CH₃),

In another embodiment, R¹ is —NR⁶⁰R⁷⁰, where R⁶⁰ is selected from —OC(O)R⁶¹, —CH₂COOH, —O-benzyl, pyridyl, and —OC(S)NR⁶²R⁶³; and R⁷⁰ is H. In still another embodiment, R¹ is —NR⁶⁰R⁷⁰, where R⁶⁰ is H or —OH, and R⁷⁰ is —C₁₋₆alkyl or —C(O)R⁷¹. In yet another embodiment, R¹ is —NR⁶⁰R⁷⁰, where R⁶⁰ is selected from —OC(O)R⁶¹, —CH₂COOH, —O-benzyl, pyridyl, and —OC(S)NR⁶²R⁶³; and R⁷⁰ is —C₁₋₆alkyl or —C(O)R⁷¹. In one aspect of the invention, these compounds may find particular utility as prodrugs or as intermediates in the synthetic procedures described herein. For example, in one embodiment, R¹ is —OR¹⁰ and R¹⁰ is —C₁₋₆alkylene-OC(O)R¹³, such as —O—CH(CH₃)OC(O)—O-cyclohexyl:

making the compound a cilexetil ester; or R¹ is —OR¹⁰ and R¹⁰ is —C₀₋₆alkylenemorpholine such as —O—(CH₂)₂-morpholine:

making the compound a 2-morpholinoethyl or mofetil ester; or R¹ is —OR¹⁰ and R¹⁰ is

such as —O—CH₂-5-methyl-[1,3]dioxol-2-one:

making the compound a medoxomil ester.

R² is selected from H and —OR²⁰. The R²⁰ moiety is H or is taken together with R¹⁰ to form —CR²¹R²²— or is taken together with R⁶⁰ to form —C(O)—; where R²¹ and R²² are independently selected from H, —C₁₋₆alkyl, and —O—C₃₋₇cycloalkyl, or R²¹ and R²² are taken together to form ═O. In one embodiment, R² is selected from H and —OR²⁰, where R²⁰ is H.

When R² is —OR²⁰ and R²⁰ is taken together with R¹⁰ to form —CR²¹R²²—, this embodiment can be depicted as:

and when R²¹ and R²² are taken together to form ═O, this embodiment can be depicted as:

When R² is —OR²⁰ and R²⁰ is taken together with R⁶⁰ to form —NHC(O)—, this embodiment can be depicted as:

In one aspect of the invention, these compounds may find particular utility as prodrugs or as intermediates in the synthetic procedures described herein.

R³ is selected from H, Cl, F, —CH₃, and —CF₃. In one embodiment, R³ is selected from H, Cl, and —CF₃.

The “X” moiety is a —C₁₋₁₀heteroaryl or a partially unsaturated —C₃₋₁₂heterocycle, with the proviso that X is not pyrazole. Compounds where X is a pyrazole are described in commonly-assigned U.S. Provisional Application No. 61/491,750 filed on May 31, 2011 and U.S. application Ser. No. 13/474,987 filed on even date herewith, both entitled “Neprilysin Inhibitors” to Gendron et al. Note that in some embodiments, R⁴, R⁵, and/or R⁶ may be absent. When present, R⁴ and/or R⁶ are on any available carbon atom. When present, R⁵ is on any available nitrogen atom. In one embodiment, X″ moiety is a —C₁₋₁₀heteroaryl, examples of which include:

pyrrole:

furan:

thiophene:

imidazole such as:

specific examples of which include:

triazole, including 1,2,3-triazole such as:

as well as 1,2,4-triazole such as:

oxazole:

specific examples of which include:

isoxazole:

specific examples of which include:

thiazole:

specific examples of which include:

isothiazole:

oxadiazole, including [1,2,4]oxadiazole such as:

as well as [1,2,3]oxadiazole such as:

and [1,3,4]oxadiazole:

thiadiazole, including [1,2,4]thiadiazole such as:

as well as [1,2,3]thiadiazole such as:

and [1,3,4]thiadiazole:

tetrazole such as:

pyridine:

specific examples of which include:

pyrazine:

pyrimidine:

specific examples of which include:

pyridazine:

benzimidazole such as:

specific examples of which include:

benzotriazole:

specific examples of which include:

benzoxazole such as:

specific examples of which include:

benzothiazole such as:

specific examples of which include:

pyridylimidazole such as:

a specific example of which includes:

pyridyltriazole such as:

specific examples of which include:

imidazopyridine such as:

a specific example of which includes:

pyrrolopyrimidine such as:

a specific example of which includes:

5-oxa-3,3a-diazacyclopenta[a]naphthalene:

In one embodiment, X is a partially unsaturated —C₂₋₁₂heterocycle, examples of which include:

dihydroimidazole:

dihydrotriazole such as:

dihydrooxazole:

dihydroisoxazole:

dihydrothiazole:

dihydroisothiazole:

dihydrooxadiazole

dihydrothiadiazole:

tetrahydropyridazine:

hexahydropyrroloquinoxaline such as

a specific example of which includes:

dihydrooxadiazabenzo[e]azulene:

In one particular embodiment, X is selected from pyrrole, furan, thiophene, imidazole, triazole, oxazole, isoxazole, thiazole, isothiazole, oxadiazole, thiadiazole, tetrazole, pyridine, pyrazine, pyrimidine, pyridazine, benzimidazole, benzotriazole, benzoxazole, benzothiazole, pyridylimidazole, pyridyltriazole, imidazopyridine, pyrrolopyrimidine, 5-oxa-3,3a-diazacyclopenta[a]naphthalene, dihydroimidazole, dihydrotriazole, dihydrooxazole, dihydroisoxazole, dihydrothiazole, dihydroisothiazole, dihydrooxadiazole, dihydrothiadiazole, tetrahydropyridazine, hexahydropyrroloquinoxaline, and dihydrooxadiazabenzo[e]azulene.

In one particular embodiment, X is selected from furan, thiophene, imidazole, triazole, oxazole, isoxazole, pyridine, pyrazine, pyrimidine, pyridazine, benzimidazole, benzotriazole, pyridylimidazole, pyridyltriazole, imidazopyridine, pyrrolopyrimidine, 5-oxa-3,3a-diazacyclopenta[a]naphthalene, dihydrotriazole, dihydroisoxazole, tetrahydropyridazine, hexahydropyrroloquinoxaline, and dihydrooxadiazabenzo[e]azulene.

The R⁴ moiety may be absent. When present, the R⁴ moiety is attached to a carbon atom and is selected from H; halo; —C₀₋₅alkylene-OH; —NH₂; —C₁₋₆alkyl; —CF₃; —C₃₋₇cycloalkyl; —C₀₋₂alkylene-O—C₁₋₆alkyl; —C(O)R⁴⁰; —C₀₋₁alkylene-C(O)OR⁴¹; —C(O)NR⁴²R⁴³; —NHC(O)R⁴⁴; ═O; —NO₂; furan; pyrazine; naphthalene; pyridine; pyrazole optionally substituted with methyl; thiophene optionally substituted with methyl; and phenyl optionally substituted with one group selected from halo, —OH, —CF₃, —OCH₃, —NHC(O)CH₃, and phenyl; and R⁴ can also be phenyl substituted with —C(O)OR⁴¹ when X is pyridazine, pyrazine, or pyridine. The R⁴⁰ moiety is H or —C₁₋₆alkyl. The R⁴¹ moiety is selected from H, —C₁₋₆alkyl, —C₁₋₃alkylene-C₆₋₁₀aryl, —C₁₋₃alkylene-C₁₋₉heteroaryl, —C₃₋₇cycloalkyl, —[(CH₂)₂O]₁₋₃CH₃, —C₁₋₆alkylene-OC(O)R¹³, —C₁₋₆alkylene-NR¹⁴R¹⁵, —C₁₋₆alkylene-C(O)R¹⁷, —C₀₋₆alkylenemorpholine, —C₁₋₆alkylene-SO₂—C₁₋₆alkyl,

R¹³ is selected from —C₁₋₆alkyl, —O—C₁₋₆alkyl, —C₃₋₇cycloalkyl, —O—C₃₋₇cycloalkyl, phenyl, —O-phenyl, —NR¹⁴R¹⁵, and —CH(NH₂)CH₂COOCH₃; R¹⁴ and R¹⁵ are independently selected from H, —C₁₋₆alkyl, and benzyl, or R¹⁴ and R¹⁵ are taken together as —(CH₂)₃₋₆—, —C(O)—(CH₂)₃—, or —(CH₂)₂O(CH₂)₂—; R¹⁶ is —C₁₋₆alkyl or —C₀₋₆alkylene-C₆₋₁₀aryl; and R¹⁷ is selected from —O—C₁₋₆alkyl, —O-benzyl, and —NR¹⁴R¹⁵. The R⁴² and R⁴³ moieties are independently selected from H, —C₁₋₆alkyl, —CH₂COOH, —(CH₂)₂OH, —(CH₂)₂OCH₃, —(CH₂)₂SO₂NH₂, —(CH₂)₂N(CH₃)₂, —C₃₋₇cycloalkyl, and —(CH₂)₂-imidazole; or R⁴² and R⁴³ are taken together to form a saturated or partially unsaturated —C₃₋₅heterocycle optionally substituted with —OH, —COOR⁴¹, or —CONH₂, and optionally containing an oxygen atom in the ring. The R⁴⁴ moiety is selected from —C₁₋₆alkyl; —C₀₋₁alkylene-O—C₁₋₆alkyl; phenyl optionally substituted with halo or —OCH₃; and —C₁₋₉heteroaryl.

In one embodiment, R⁴ is selected from H; halo (e.g. chloro, fluoro, and bromo); —C₀₋₅alkylene-OH (e.g., —OH); —C₁₋₆alkyl (e.g., —CH₃, —CH₂CH₃, —(CH₂)₂CH₃, and —(CH₂)₃CH₃); —C₀₋₁alkylene-C(O)OR⁴¹ (e.g., —COOR⁴¹); ═O; phenyl optionally substituted with one halo (e.g., chloro); and phenyl substituted with —C(O)OR⁴¹ when X is pyridazine, pyrazine, or pyridine; and R⁴¹ is selected from H and —C₁₋₆alkyl (e.g., —CH₂CH₃).

In one particular embodiment, R⁴ is —C₀₋₁alkylene-C(O)OR⁴¹, where R⁴¹ is H. In another particular embodiment, R⁴ is —C₀₋₁alkylene-C(O)OR⁴¹, where R⁴¹ is selected from —C₁₋₆alkyl, —C₁₋₃alkylene-C₆₋₁₀aryl, —C₁₋₃alkylene-C₁₋₉heteroaryl, —C₃₋₇cycloalkyl, —[(CH₂)₂O]₁₋₃CH₃, —C₁₋₆alkylene-OC(O)R¹³, —C₁₋₆alkylene-NR¹⁴R¹⁵, —C₁₋₆alkylene-C(O)R¹⁷, —C₀₋₆alkylenemorpholine, —C₁₋₆alkylene-SO₂—C₁₋₆alkyl,

In one aspect of the invention, these latter compounds may find particular utility as prodrugs or as intermediates in the synthetic procedures described herein.

The R⁵ moiety may be absent. When present, the R⁵ moiety is attached to a nitrogen atom and is selected from H; —C₁₋₆alkyl; —C₀₋₃alkylene-OH; —[(CH₂)₂O]₁₋₃CH₃; —C₁₋₃alkylene-C(O)OR⁵⁰; —CH₂—C(O)NR⁵¹R⁵²; —C₀₋₂alkylene-pyridine optionally substituted with halo; —CH₂-isoxazole optionally substituted with methyl; —CH₂-pyrimidine optionally substituted with —O—C₁₋₆alkyl; —C₂alkylene-phenyl;

The R⁵⁰ moiety is selected from H, —C₁₋₆alkyl, —C₁₋₃alkylene-C₆₋₁₀aryl, —C₁₋₃alkylene-C₁₋₉heteroaryl, —C₃₋₇cycloalkyl, —[(CH₂)₂O]₁₋₃CH₃, —C₁₋₆alkylene-OC(O)R¹³, —C₁₋₆alkylene-NR¹⁴R¹⁵, —C₁₋₆alkylene-C(O)R¹⁷, —C₀₋₆alkylenemorpholine, —C₁₋₆alkylene-SO₂—C₁₋₆alkyl,

where R¹³ is selected from —C₁₋₆alkyl, —O—C₁₋₆alkyl, —C₃₋₇cycloalkyl, —O—C₃₋₇cycloalkyl, phenyl, —O-phenyl, —NR¹⁴R¹⁵, and —CH(NH₂)CH₂COOCH₃; R¹⁴ and R¹⁵ are independently selected from H, —C₁₋₆alkyl, and benzyl, or R¹⁴ and R¹⁵ are taken together as —(CH₂)₃₋₆—, —C(O)—(CH₂)₃—, or —(CH₂)₂—O(CH₂)₂—; R¹⁶ is —C₁₋₆alkyl or —C₀₋₆alkylene-C₆₋₁₀aryl; and R¹⁷ is selected from —O—C₁₋₆alkyl, —O-benzyl, and —NR¹⁴R¹⁵. The R⁵¹ and R⁵² moieties are independently selected from H, —C₁₋₆alkyl, —CH₂COOH, —(CH₂)₂OH, —(CH₂)₂OCH₃, —(CH₂)₂SO₂NH₂, —(CH₂)₂N(CH₃)₂, —C₃₋₇cycloalkyl, and —(CH₂)₂-imidazole; or R⁵² and R⁵³ are taken together to form a saturated or partially unsaturated —C₃₋₅heterocycle optionally substituted with —OH, —COOR⁴¹, or —CONH₂, and optionally containing an oxygen atom in the ring. The R⁵³, R⁵⁴, R⁵⁵, R⁵⁶, and R⁵⁷ moieties are independently selected from H, halo, —C₁₋₆alkyl, —O—C₁₋₆alkyl, and —S—C₁₋₆alkyl, where each —C₁₋₆alkyl is optionally substituted with 1-5 fluoro atoms.

In one embodiment, R⁵ is absent or is selected from H; —C₀₋₃alkylene-OH (e.g., —OH and —(CH₂)₃OH); —C₁₋₃alkylene-C(O)OR⁵⁰ (e.g., —CH₂C(O)OR⁵⁰); —CH₂—C(O)NR⁵¹R⁵²; —C₀₋₂alkylene-pyridine optionally substituted with halo (e.g., bromo), for example:

where R⁵⁰ is H; R⁵¹ and R⁵² are H; R⁵³ is selected from H, halo (e.g., fluoro), and —O—C₁₋₆alkyl (e.g., —OCH₃); R⁵⁴ is selected from H and halo (e.g., fluoro); R⁵⁵ is selected from H, halo (e.g., chloro), and —O—C₁₋₆alkyl (e.g., —OCH₃); R⁵⁶ is H; and R⁵⁷ is H.

In one particular embodiment, R⁵ is —C₁₋₃alkylene-C(O)OR⁵⁰, where R⁵⁰ is H. In another particular embodiment, R⁵ is —C₁₋₃alkylene-C(O)OR⁵⁰, where R⁵⁰ is selected from —C₁₋₆alkyl, —C₁₋₃alkylene-C₆₋₁₀aryl, —C₁₋₃alkylene-C₁₋₉heteroaryl, —C₃₋₇cycloalkyl, —[(CH₂)₂O]₁₋₃CH₃, —C₁₋₆alkylene-OC(O)R¹³, —C₁₋₆alkylene-NR¹⁴R¹⁵, —C₁₋₆alkylene-C(O)R¹⁷, —C₀₋₆alkylenemorpholine, —C₁₋₆alkylene-SO₂—C₁₋₆alkyl,

In one aspect of the invention, these latter compounds may find particular utility as prodrugs or as intermediates in the synthetic procedures described herein.

The R⁶ may be absent. When present, the R⁶ moiety is attached to a carbon atom and is selected from H, halo, —OH, —C₁₋₆alkyl, and —O—C₁₋₆alkyl. In one embodiment, R⁶ is absent or is selected from H, halo (e.g., chloro and fluoro), —OH, and —C₁₋₆alkyl (e.g., —CH₃).

Specific examples of compounds of formula I include compounds of formulas Ic to Il, as described below. In one embodiment R² is —OR²⁰, R²⁰ is H, and R³ is Cl, which can be depicted as formula Ic:

In an exemplary embodiment of compounds of formula Ic, R¹ is —OR¹⁰; R¹⁰ is H or —C₁₋₆alkyl; X is selected from furan, triazole, oxazole, isoxazole, pyridine, pyrazine, pyrimidine, benzotriazole, pyridylimidazole, pyridyltriazole, imidazopyridine, 5-oxa-3,3a-diazacyclopenta[a]naphthalene, tetrahydropyridazine, and dihydrooxadiazabenzo[e]azulene; R⁴ is selected from H; halo; —C₀₋₅alkylene-OH; —C₁₋₆alkyl; —C₀₋₁alkylene-C(O)OR⁴¹; ═O; phenyl optionally substituted with one halo, and phenyl substituted with —C(O)OR⁴¹ when X is pyrazine or pyridine; R⁴¹ is H or —C₁₋₆alkyl; R⁵ is absent or is selected from H, —C₀₋₃alkylene-OH,

R⁵³ is H or halo; R⁵⁴ is H or halo; R⁵⁵ is selected from H, halo, and —O—C₁₋₆alkyl; R⁵⁶ is H; R⁵⁷ is H; and R⁶ is absent or is selected from H, halo, —OH, and —C₁₋₆alkyl.

In another embodiment R² is —OR²⁰, R²⁰ is H; and R³ is —CF₃, which can be depicted as formula Id:

In an exemplary embodiment of compounds of formula Id, R¹ is —OR¹⁰; R¹⁰ is H or —C₁₋₆alkyl; X is pyridyltriazole; R⁴ is H; R⁵ is H; and R⁶ is H.

In still another embodiment R² is —OR²⁰; R²⁰ is H; and R³ is H, which can be depicted as formula Ie:

In an exemplary embodiment of compounds of formula Ie, R¹ is —OR¹⁰; R¹⁰ is H or —C₁₋₆alkyl; X is selected from thiophene, imidazole, pyridine, pyrazine, pyridazine, benzimidazole, benzotriazole, pyridyltriazole, pyrrolopyrimidine, and hexahydropyrroloquinoxaline; R⁴ is selected from H; halo; —C₁₋₆alkyl; —C₀₋₁alkylene-C(O)OR⁴¹; ═O; and phenyl substituted with —C(O)OR⁴¹ when X is pyridazine, pyrazine, or pyridine; R⁴¹ is H or —C₁₋₆alkyl; R⁵ is absent or is selected from H; —C₀₋₃alkylene-OH; —C₁₋₃alkylene-C(O)OR⁵⁰; —CH₂—C(O)NR⁵¹R⁵²; —C₀₋₂alkylene-pyridine optionally substituted with halo;

R⁵⁰ is H; R⁵¹ and R⁵² are H; R⁵³ is H or —O—C₁₋₆alkyl; R⁵⁴ is H; R⁵⁵ is H or —O—C₁₋₆alkyl; R⁵⁶ is H; R⁵⁷ is H; and R⁶ is absent or is selected from H and halo.

In one embodiment R² is —OR²⁰, R²⁰ is H, and R³ is —CH₃, which can be depicted as formula If:

In another embodiment R² is —OR²⁰, R²⁰ is H and R³ is F, which can be depicted as formula Ig:

In one embodiment R² is H and R³ is Cl, which can be depicted as formula Ih:

In an exemplary embodiment of compounds of formula Ih, R¹ is —OR¹⁰; R¹⁰ is H or —C₁₋₆alkyl; X is selected from triazole, pyridine, pyrimidine, pyridazine, benzotriazole, and pyridyltriazole; R⁴ is selected from H, halo, —C₀₋₅alkylene-OH, —C₁₋₆alkyl, and phenyl substituted with —C(O)OR⁴¹ when X is pyridazine or pyridine; R⁴¹ is H; R⁵ is absent or is H; and R⁶ is absent or is H.

In another embodiment R² is H and R³ is —CF₃, which can be depicted as formula Ii:

In still another embodiment R² is H and R³ is H, which can be depicted as formula Ij:

In one embodiment R² is H and R³ is —CH₃, which can be depicted as formula Ik:

In another embodiment R² is H and R³ is F, which can be depicted as formula Il:

In yet another embodiment, R¹ is —OR¹⁰; R¹⁰ is H or —C₁₋₆alkyl; X is selected from thiophene, imidazole, triazole, oxazole, isoxazole, pyridine, pyrazine, pyrimidine, pyridazine, benzimidazole, benzotriazole, pyridylimidazole, pyridyltriazole, imidazopyridine, pyrrolopyrimidine, 5-oxa-3,3a-diazacyclopenta[a]naphthalene, dihydrotriazole, dihydroisoxazole, tetrahydropyridazine, hexahydropyrroloquinoxaline, and dihydrooxadiazabenzo[e]azulene; R⁴ is selected from H; halo; —C₀₋₅alkylene-OH; —C₁₋₆alkyl; —C₀₋₁alkylene-C(O)OR⁴¹; ═O; phenyl optionally substituted with one halo; and phenyl substituted with —C(O)OR⁴¹ when X is pyridazine, pyrazine, or pyridine; R⁴¹ is selected from H and —C₁₋₆alkyl; R⁵ is absent or is selected from H; —C₀₋₃alkylene-OH; —C₁₋₃alkylene-C(O)OR⁵⁰; —CH₂—C(O)NR⁵¹R⁵²; —C₀₋₂alkylene-pyridine optionally substituted with halo;

R⁵⁰ is H; R⁵¹ and R⁵² are H; R⁵³ is selected from H, halo, and —O—C₁₋₆alkyl; R⁵⁴ is H or halo; R⁵⁵ is selected from H, halo, and —O—C₁₋₆alkyl; R⁵⁶ is H; R⁵⁷ is H; and R⁶ is absent or is selected from H, halo, —OH, and —C₁₋₆alkyl. In one embodiment, these compounds have formulas Ic to Il. In still another compound, these compounds have R² being selected from H and —OR²⁰, where R²⁰ is H; and R³ being selected from H, Cl, and —CF₃.

In addition, particular compounds of formula I that are of interest include those set forth in the Examples below, as well as pharmaceutically acceptable salts thereof.

General Synthetic Procedures

Compounds of the invention can be prepared from readily available starting materials using the following general methods, the procedures set forth in the Examples, or by using other methods, reagents, and starting materials that are known to those of ordinary skill in the art. Although the following procedures may illustrate a particular embodiment of the invention, it is understood that other embodiments of the invention can be similarly prepared using the same or similar methods or by using other methods, reagents and starting materials known to those of ordinary skill in the art. It will also be appreciated that where typical or preferred process conditions (for example, reaction temperatures, times, mole ratios of reactants, solvents, pressures, etc.) are given, other process conditions can also be used unless otherwise stated. In some instances, reactions were conducted at room temperature and no actual temperature measurement was taken. It is understood that room temperature can be taken to mean a temperature within the range commonly associated with the ambient temperature in a laboratory environment, and will typically be in the range of about 18° C. to about 30° C. In other instances, reactions were conducted at room temperature and the temperature was actually measured and recorded. While optimum reaction conditions will typically vary depending on various reaction parameters such as the particular reactants, solvents and quantities used, those of ordinary skill in the art can readily determine suitable reaction conditions using routine optimization procedures.

Additionally, as will be apparent to those skilled in the art, conventional protecting groups may be necessary or desired to prevent certain functional groups from undergoing undesired reactions. The choice of a suitable protecting group for a particular functional group as well as suitable conditions and reagents for protection and deprotection of such functional groups are well-known in the art. Protecting groups other than those illustrated in the procedures described herein may be used, if desired. For example, numerous protecting groups, and their introduction and removal, are described in T. W. Greene and G. M. Wuts, Protecting Groups in Organic Synthesis, Fourth Edition, Wiley, New York, 2006, and references cited therein. More specifically, the following abbreviations and reagents are used in the schemes presented below:

P¹ represents a “carboxy-protecting group,” a term used herein to mean a protecting group suitable for preventing undesired reactions at a carboxy group. Representative carboxy-protecting groups include, but are not limited to, methyl, ethyl, t-butyl, benzyl (Bn), p-methoxybenzyl (PMB), 9-fluorenylmethyl (Fm), trimethylsilyl (TMS), t-butyldimethylsilyl (TBDMS), diphenylmethyl (benzhydryl, DPM) and the like. Standard deprotection techniques and reagents are used to remove the P¹ group, and may vary depending upon which group is used. For example, sodium or lithium hydroxide is commonly used when P¹ is methyl, an acid such as TFA or HCl is commonly used when P¹ is ethyl or t-butyl, and H₂/Pd/C may be used when P¹ is benzyl.

P² represents an “amino-protecting group,” a term used herein to mean a protecting group suitable for preventing undesired reactions at an amino group. Representative amino-protecting groups include, but are not limited to, t-butoxycarbonyl (BOC), trityl (Tr), benzyloxycarbonyl (Cbz), 9-fluorenylmethoxycarbonyl (Fmoc), formyl, trimethylsilyl (TMS), t-butyldimethylsilyl (TBDMS), and the like. Standard deprotection techniques are used to remove the P² group. For example, a BOC group can be removed using an acidic reagent such as TFA in DCM or HCl in 1,4-dioxane, while a Cbz group can be removed by employing catalytic hydrogenation conditions such as H₂ (1 atm) and 10% Pd/C in an alcoholic solvent (“H₂/Pd/C”).

P³ represents a “hydroxyl-protecting group,” a term that is used herein to mean a protecting group suitable for preventing undesired reactions at a hydroxyl group. Representative hydroxyl-protecting groups include, but are not limited to C₁₋₆alkyls, silyl groups including triC₁₋₆alkylsilyl groups, such as trimethylsilyl (TMS), triethylsilyl (TES), and tert-butyldimethylsilyl (TBDMS); esters (acyl groups) including C₁₋₆alkanoyl groups, such as formyl, acetyl, and pivaloyl, and aromatic acyl groups such as benzoyl; arylmethyl groups such as benzyl (Bn), p-methoxybenzyl (PMB), 9-fluorenylmethyl (Fm), and diphenylmethyl (benzhydryl, DPM); and the like. Standard deprotection techniques and reagents are used to remove the P³ group, and may vary depending upon which group is used. For example, H₂/Pd/C is commonly used when P³ is benzyl, while NaOH is commonly used when P³ is an acyl group.

Suitable bases for use in these schemes include, by way of illustration and not limitation, potassium carbonate, calcium carbonate, sodium carbonate, triethylamine, pyridine, 1,8-diazabicyclo-[5.4.0]undec-7-ene (DBU), N,N-diisopropylethylamine (DIPEA), 4-methylmorpholine, sodium hydroxide, potassium hydroxide, potassium t-butoxide, and metal hydrides.

Suitable inert diluents or solvents for use in these schemes include, by way of illustration and not limitation, tetrahydrofuran (THF), acetonitrile (MeCN), N,N-dimethylformamide (DMF), N,N-dimethylacetamide (DMA), dimethyl sulfoxide (DMSO), toluene, dichloromethane (DCM), chloroform (CHCl₃), carbon tetrachloride (CCl₄), 1,4-dioxane, methanol, ethanol, water, and the like.

Suitable carboxylic acid/amine coupling reagents include benzotriazol-1-yloxytris(dimethylamino)phosphonium hexafluorophosphate (BOP), benzotriazol-1-yloxytripyrrolidinophosphonium hexafluorophosphate (PyBOP), N,N,N′,N′-tetramethyl-O-(7-azabenzotriazol-1-yl)uronium hexafluorophosphate (HATU), 1,3-dicyclohexylcarbodiimide (DCC), N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide (EDCI), carbonyldiimidazole (CDI), 1-hydroxybenzotriazole (HOBt), and the like. Coupling reactions are conducted in an inert diluent in the presence of a base such as DIPEA, and are performed under conventional amide bond-forming conditions.

All reactions are typically conducted at a temperature within the range of about −78 C to 100° C., for example at room temperature. Reactions may be monitored by use of thin layer chromatography (TLC), high performance liquid chromatography (HPLC), and/or LCMS until completion. Reactions may be complete in minutes, or may take hours, typically from 1-2 hours and up to 48 hours. Upon completion, the resulting mixture or reaction product may be further treated in order to obtain the desired product. For example, the resulting mixture or reaction product may be subjected to one or more of the following procedures: concentrating or partitioning (for example, between EtOAc and water or between 5% THF in EtOAc and 1M phosphoric acid); extraction (for example, with EtOAc, CHCl₃, DCM, chloroform); washing (for example, with saturated aqueous NaCl, saturated NaHCO₃, Na₂CO₃ (5%), CHCl₃ or 1M NaOH); drying (for example, over MgSO₄, over Na₂SO₄, or in vacuo); filtering; crystallizing (for example, from EtOAc and hexanes); being concentrated (for example, in vacuo); and/or purification (e.g., silica gel chromatography, flash chromatography, preparative HPLC, reverse phase-HPLC, or crystallization).

Compounds of formula I, as well as their salts, can be prepared as shown in Scheme I:

Compound 1 is coupled with compound 2. In instances where R¹ is a group such as —OCH₃ or —OCH₂CH₃, the coupling step may be followed by a deprotection step to provide a compound of formula I where R¹ is a group such as —OH. Thus, one method of preparing compounds of the invention involves coupling compound 1 and compound 2, with an optional deprotection step to form a compound of formula I or a pharmaceutically acceptable salt thereof.

Compounds of formula I and their salts, where R⁴ is optionally substituted phenyl, can also be prepared as shown in Scheme II:

Again, as with Scheme I, this is a standard coupling reaction between compound 1 and compound 2a to yield the compound 3, where L a leaving group such as halo (e.g., bromo and chloro). Compound 3 is then reacted with optionally substituted 4,4,5,5-tetramethyl-2-phenyl-[1,3,2]dioxaborolane in a palladium-catalyzed coupling reaction. To prepare a compound of formula I where R⁴ is unsubstituted phenyl, then R is hydrogen. Otherwise, R is selected from halo, —OH, —CF₃, —OCH₃, —NHC(O)CH₃, and phenyl. R may also be —C(O)OR⁴¹ when X is pyridazine, pyrazine, or pyridine. Exemplary substituted dioxaborolanes include 2-t-butoxycarbonylphenylboronic acid pinacol ester.

The palladium-catalyzed coupling reaction is typically conducted in an inert diluent in the presence of a suitable base such as potassium carbonate or sodium carbonate, and may be followed by a deprotection step to provide a compound of formula I where R¹ is a group such as —OH. Thus, another method of preparing compounds of the invention involves coupling compound 1 and compound 2a to form compound 3, coupling compound 3 with optionally substituted 4,4,5,5-tetramethyl-2-phenyl-[1,3,2]dioxaborolane, and an optional deprotection step to form a compound of formula I or a pharmaceutically acceptable salt thereof.

Compounds of formula I and their salts, where R⁴ is optionally substituted phenyl, can also be prepared as shown in Scheme III:

As with Scheme I, the first step is a palladium-catalyzed coupling reaction between compound 2b and an optionally substituted 4,4,5,5-tetramethyl-2-phenyl-[1,3,2]dioxaborolane to form compound 4, where L a leaving group such as halo (e.g., bromo and chloro) and P¹ is a carboxy-protecting group selected from methyl, ethyl, t-butyl, benzyl, p-methoxybenzyl, 9-fluorenylmethyl, trimethylsilyl, t-butyldimethylsilyl, and diphenylmethyl. Compound 4 is then coupled with compound 1, followed by an optional deprotection step to provide a compound of formula I where R¹ is a group such as —OH. Thus, another method of preparing compounds of the invention involves coupling compound 2b with optionally substituted 4,4,5,5-tetramethyl-2-phenyl-[1,3,2]dioxaborolane to form compound 4, coupling compound 4 and compound 1, and an optional deprotection step to form a compound of formula I or a pharmaceutically acceptable salt thereof.

Compounds 1, 2, 2a, and 2b are generally commercially available or can be prepared using procedures that are known in the art as well as those that are set forth in the Examples herein.

Certain intermediates described herein are believed to be novel and accordingly, such compounds are provided as further aspects of the invention including, for example, the compounds of formula II, or a salt thereof:

where P is selected from —O—P¹, —NHP², and —NH(O—P³); P¹ is a carboxy-protecting group selected from methyl, ethyl, t-butyl, benzyl, p-methoxybenzyl, 9-fluorenylmethyl, trimethylsilyl, t-butyldimethylsilyl, and diphenylmethyl; P² is an amino-protecting group selected from t-butoxycarbonyl, trityl, benzyloxycarbonyl, 9-fluorenylmethoxycarbonyl, formyl, trimethylsilyl, and t-butyldimethylsilyl; and P³ is a hydroxyl-protecting group selected from C₁₋₆alkyl, silyl groups, esters, and arylmethyl groups; and R²—R⁶ are as defined for formula I. Thus, another method of preparing compounds of the invention involves deprotecting a compound of formula II.

Further details regarding specific reaction conditions and other procedures for preparing representative compounds of the invention or intermediates thereof are described in the Examples set forth below.

Utility

Compounds of the invention possess neprilysin (NEP) inhibition activity, that is, the compounds are able to inhibit enzyme-catalytic activity. In another embodiment, the compounds do not exhibit significant inhibitory activity of the angiotensin-converting enzyme. One measure of the ability of a compound to inhibit NEP activity is the inhibition constant (pK_(i)). The pK_(i) value is the negative logarithm to base 10 of the dissociation constant (K_(i)), which is typically reported in molar units. Compounds of the invention of particular interest are those having a pK_(i) at NEP greater than or equal to 6.0, particularly those having a pK_(i) greater than or equal to 7.0, and even more particularly those having a pK_(i) greater than or equal to 8.0. In one embodiment, compounds of interest have a pK_(i) in the range of 6.0-6.9; in another embodiment, compounds of interest have a pK_(i) in the range of 7.0-7.9; in yet another embodiment, compounds of interest have a pK_(i) in the range of 8.0-8.9; and in still another embodiment, compounds of interest have a pK_(i) in the range of greater than or equal to 9.0. Such values can be determined by techniques that are well known in the art, as well as in the assays described herein.

Another measure of the ability of a compound to inhibit NEP activity is the apparent inhibition constant (IC₅₀), which is the molar concentration of compound that results in half-maximal inhibition of substrate conversion by the NEP enzyme. The pIC₅₀ value is the negative logarithm to base 10 of the IC₅₀. Compounds of the invention that are of particular interest, include those that exhibit a pIC₅₀ for NEP greater than or equal to about 5.0. Compounds of interest also include those having a pIC₅₀ for NEP≧about 6.0 or a pIC₅₀ for NEP≧about 7.0. In another embodiment, compounds of interest have a pIC₅₀ for NEP within the range of about 7.0-11.0; and in another embodiment, within the range of about 8.0-11.0, such as within the range of about 8.0-10.0.

It is noted that in some cases, compounds of the invention may possess weak NEP inhibition activity. In such cases, those of skill in the art will recognize that these compounds still have utility as research tools.

Exemplary assays to determine properties of compounds of the invention, such as the NEP inhibiting activity, are described in the Examples and include by way of illustration and not limitation, assays that measure NEP inhibition (described in Assay 1). Useful secondary assays include assays to measure ACE inhibition (also described in Assay 1) and aminopeptidase P (APP) inhibition (described in Sulpizio et al. (2005) JPET 315:1306-1313). A pharmacodynamic assay to assess the in vivo inhibitory potencies for ACE and NEP in anesthetized rats is described in Assay 2 (see also Seymour et al. (1985) Hypertension 7(Suppl I):I-35-I-42 and Wigle et al. (1992) Can. J. Physiol. Pharmacol. 70:1525-1528), where ACE inhibition is measured as the percent inhibition of the angiotensin I pressor response and NEP inhibition is measured as increased urinary cyclic guanosine 3′,5′-monophosphate (cGMP) output.

There are many in vivo assays that can be used to ascertain further utilities of the compounds of the invention. The conscious spontaneously hypertensive rat (SHR) model is a renin dependent hypertension model, and is described in Assay 3. See also Intengan et al. (1999) Circulation 100(22):2267-2275 and Badyal et al. (2003) Indian Journal of Pharmacology 35:349-362. The conscious desoxycorticosterone acetate-salt (DOCA-salt) rat model is a volume dependent hypertension model that is useful for measuring NEP activity, and is described in Assay 4. See also Trapani et al. (1989) J. Cardiovasc. Pharmacol. 14:419-424, Intengan et al. (1999) Hypertension 34(4):907-913, and Badyal et al. (2003) supra). The DOCA-salt model is particularly useful for evaluating the ability of a test compound to reduce blood pressure as well as to measure a test compound's ability to prevent or delay a rise in blood pressure. The Dahl salt-sensive (DSS) hypertensive rat model is a model of hypertension that is sensitive to dietary salt (NaCl), and is described in Assay 5. See also Rapp (1982) Hypertension 4:753-763. The rat monocrotaline model of pulmonary arterial hypertension described, for example, in Kato et al. (2008) J. Cardiovasc. Pharmacol. 51(1):18-23, is a reliable predictor of clinical efficacy for the treatment of pulmonary arterial hypertension. Heart failure animal models include the DSS rat model for heart failure and the aorto-caval fistula model (AV shunt), the latter of which is described, for example, in Norling et al. (1996) J. Amer. Soc. Nephrol. 7:1038-1044. Other animal models, such as the hot plate, tail-flick and formalin tests, can be used to measure the analgesic properties of compounds of the invention, as well as the spinal nerve ligation (SNL) model of neuropathic pain. See, for example, Malmberg et al. (1999) Current Protocols in Neuroscience 8.9.1-8.9.15.

Compounds of the invention are expected to inhibit the NEP enzyme in any of the assays listed above, or assays of a similar nature. Thus, the aforementioned assays are useful in determining the therapeutic utility of compounds of the invention, for example, their utility as antihypertensive agents or antidiarrheal agents. Other properties and utilities of compounds of the invention can be demonstrated using other in vitro and in vivo assays well-known to those skilled in the art. Compounds of formula I may be active drugs as well as prodrugs. Thus, when discussing the activity of compounds of the invention, it is understood that any such prodrugs may not exhibit the expected activity in an assay, but are expected to exhibit the desired activity once metabolized.

Compounds of the invention are expected to be useful for the treatment and/or prevention of medical conditions responsive to NEP inhibition. Thus it is expected that patients suffering from a disease or disorder that is treated by inhibiting the NEP enzyme or by increasing the levels of its peptide substrates, can be treated by administering a therapeutically effective amount of a compound of the invention. For example, by inhibiting NEP, the compounds are expected to potentiate the biological effects of endogenous peptides that are metabolized by NEP, such as the natriuretic peptides, bombesin, bradykinins, calcitonin, endothelins, enkephalins, neurotensin, substance P and vasoactive intestinal peptide. Thus, these compounds are expected to have other physiological actions, for example, on the renal, central nervous, reproductive and gastrointestinal systems.

In one embodiment of the invention, patients suffering from a disease or disorder that is treated by inhibiting the NEP enzyme, are treated by administering a compound of the invention that is in its active form, i.e., a compound of formula I where R¹ is selected from —OR¹⁰ and —NR⁶⁰R⁷⁰, where R¹⁰ is H, R⁶⁰ is H or —OH, and R⁷⁰ is H; and R², R³, X, R⁴, R⁵, and R⁶ are as defined for formula I.

In another embodiment, patients are treated by administering a compound that is metabolized in vitro to form a compound of formula I, where R¹ is —OR¹⁰ or —NR⁶⁰R⁷⁰; R¹⁰ is H, R⁶⁰ is H or —OH, R⁷⁰ is H; and R², R³, X, R⁴, R⁵, and R⁶ are as defined for formula I.

In another embodiment, patients are treated by administering a compound of the invention that is in its prodrug form at the R¹ group, i.e., a compound of formula I where:

R¹ is —OR¹⁰; and R¹⁰ is selected from —C₁₋₆alkyl, —C₁₋₃alkylene-C₆₋₁₀aryl, —C₁₋₃alkylene-C₁₋₉heteroaryl, —C₃₋₇cycloalkyl, —[(CH₂)₂O]₁₋₃CH₃, —C₁₋₆alkylene-OC(O)R¹³, —C₁₋₆alkylene-NR¹⁴R¹⁵, —C₁₋₆alkylene-C(O)R¹⁷, —C₀₋₆alkylenemorpholine, —C₁₋₆alkylene-SO₂—C₁₋₆alkyl,

R¹ is —NR⁶⁰R⁷⁰; R⁶⁰ is selected from —OC(O)R⁶¹, —CH₂COOH, —O-benzyl, pyridyl, and —OC(S)NR⁶²R⁶³; and R⁷⁰ is H; or

R¹ is —NR⁶⁰R⁷⁰; R⁶⁰ is selected from —OC(O)R⁶¹, —CH₂COOH, —O-benzyl, pyridyl, and —OC(S)NR⁶²R⁶³; and R⁷⁰ is —C₁₋₆alkyl or —C(O)R⁷¹; or

R¹ is —NR⁶⁰R⁷⁰; R⁶⁰ is H or —OH; and R⁷⁰ is —C₁₋₆alkyl or —C(O)R⁷¹; or

R¹ is —OR¹⁰; R² is —OR²⁰; and R²⁰ is taken together with R¹⁰ to form —CR²¹R²²-; or

R¹ is —NR⁶⁰R⁷⁰; R² is —OR²⁰; and R²⁰ is taken together with R⁶⁰ to form —C(O)—; and R², R³, X, R⁴, R⁵, R⁶, R¹³, R¹⁴, R¹⁵, R¹⁶, R¹⁷, R²¹, R²², R⁶¹, R⁶², R⁶³, and R⁷¹ are as defined for formula I.

Cardiovascular Diseases

By potentiating the effects of vasoactive peptides like the natriuretic peptides and bradykinin, compounds of the invention are expected to find utility in treating and/or preventing medical conditions such as cardiovascular diseases. See, for example, Rogues et al. (1993) Pharmacol. Rev. 45:87-146 and Dempsey et al. (2009) Amer. J. of Pathology 174(3):782-796. Cardiovascular diseases of particular interest include hypertension and heart failure. Hypertension includes, by way of illustration and not limitation: primary hypertension, which is also referred to as essential hypertension or idiopathic hypertension; secondary hypertension; hypertension with accompanying renal disease; severe hypertension with or without accompanying renal disease; pulmonary hypertension, including pulmonary arterial hypertension; and resistant hypertension. Heart failure includes, by way of illustration and not limitation: congestive heart failure; acute heart failure; chronic heart failure, for example with reduced left ventricular ejection fraction (also referred to as systolic heart failure) or with preserved left ventricular ejection fraction (also referred to as diastolic heart failure); and acute and chronic decompensated heart failure, with or without accompanying renal disease. Thus, one embodiment of the invention relates to a method for treating hypertension, particularly primary hypertension or pulmonary arterial hypertension, comprising administering to a patient a therapeutically effective amount of a compound of the invention.

For treatment of primary hypertension, the therapeutically effective amount is typically the amount that is sufficient to lower the patient's blood pressure. This would include both mild-to-moderate hypertension and severe hypertension. When used to treat hypertension, the compound may be administered in combination with other therapeutic agents such as aldosterone antagonists, aldosterone synthase inhibitors, angiotensin-converting enzyme inhibitors and dual-acting angiotensin-converting enzyme/neprilysin inhibitors, angiotensin-converting enzyme 2 (ACE2) activators and stimulators, angiotensin-II vaccines, anti-diabetic agents, anti-lipid agents, anti-thrombotic agents, AT₁ receptor antagonists and dual-acting AT₁ receptor antagonist/neprilysin inhibitors, β₁-adrenergic receptor antagonists, dual-acting β-adrenergic receptor antagonist/α₁-receptor antagonists, calcium channel blockers, diuretics, endothelin receptor antagonists, endothelin converting enzyme inhibitors, neprilysin inhibitors, natriuretic peptides and their analogs, natriuretic peptide clearance receptor antagonists, nitric oxide donors, non-steroidal anti-inflammatory agents, phosphodiesterase inhibitors (specifically PDE-V inhibitors), prostaglandin receptor agonists, renin inhibitors, soluble guanylate cyclase stimulators and activators, and combinations thereof. In one particular embodiment of the invention, a compound of the invention is combined with an AT₁ receptor antagonist, a calcium channel blocker, a diuretic, or a combination thereof, and used to treat primary hypertension. In another particular embodiment of the invention, a compound of the invention is combined with an AT₁ receptor antagonist, and used to treat hypertension with accompanying renal disease. When used to treat resistant hypertension, the compound may be administered in combination with other therapeutic agents such as aldosterone synthase inhibitors.

For treatment of pulmonary arterial hypertension, the therapeutically effective amount is typically the amount that is sufficient to lower the pulmonary vascular resistance. Other goals of therapy are to improve a patient's exercise capacity. For example, in a clinical setting, the therapeutically effective amount can be the amount that improves a patient's ability to walk comfortably for a period of 6 minutes (covering a distance of approximately 20-40 meters). When used to treat pulmonary arterial hypertension the compound may be administered in combination with other therapeutic agents such as α-adrenergic receptor antagonists, β₁-adrenergic receptor antagonists, β₂-adrenergic receptor agonists, angiotensin-converting enzyme inhibitors, anticoagulants, calcium channel blockers, diuretics, endothelin receptor antagonists, PDE-V inhibitors, prostaglandin analogs, selective serotonin reuptake inhibitors, and combinations thereof. In one particular embodiment of the invention, a compound of the invention is combined with a PDE-V inhibitor or a selective serotonin reuptake inhibitor and used to treat pulmonary arterial hypertension.

Another embodiment of the invention relates to a method for treating heart failure, in particular congestive heart failure (including both systolic and diastolic congestive heart failure), comprising administering to a patient a therapeutically effective amount of a compound of the invention. Typically, the therapeutically effective amount is the amount that is sufficient to lower blood pressure and/or improve renal functions. In a clinical setting, the therapeutically effective amount can be the amount that is sufficient to improve cardiac hemodynamics, like for instance reduction in wedge pressure, right atrial pressure, filling pressure, and vascular resistance. In one embodiment, the compound is administered as an intravenous dosage form. When used to treat heart failure, the compound may be administered in combination with other therapeutic agents such as adenosine receptor antagonists, advanced glycation end product breakers, aldosterone antagonists, AT₁ receptor antagonists, β₁-adrenergic receptor antagonists, dual-acting β-adrenergic receptor antagonist/α₁-receptor antagonists, chymase inhibitors, digoxin, diuretics, endothelin converting enzyme (ECE) inhibitors, endothelin receptor antagonists, natriuretic peptides and their analogs, natriuretic peptide clearance receptor antagonists, nitric oxide donors, prostaglandin analogs, PDE-V inhibitors, soluble guanylate cyclase activators and stimulators, and vasopressin receptor antagonists. In one particular embodiment of the invention, a compound of the invention is combined with an aldosterone antagonist, a β₁-adrenergic receptor antagonist, an AT₁ receptor antagonist, or a diuretic, and used to treat congestive heart failure.

Diarrhea

As NEP inhibitors, compounds of the invention are expected to inhibit the degradation of endogenous enkephalins and thus such compounds may also find utility for the treatment of diarrhea, including infectious and secretory/watery diarrhea. See, for example, Baumer et al. (1992) Gut 33:753-758; Farthing (2006) Digestive Diseases 24:47-58; and Marçais-Collado (1987) Eur. J. Pharmacol. 144(2):125-132. When used to treat diarrhea, compounds of the invention may be combined with one or more additional antidiarrheal agents.

Renal Diseases

By potentiating the effects of vasoactive peptides like the natriuretic peptides and bradykinin, compounds of the invention are expected to enhance renal function (see Chen et al. (1999) Circulation 100:2443-2448; Lipkin et al. (1997) Kidney Int. 52:792-801; and Dussaule et al. (1993) Clin. Sci. 84:31-39) and find utility in treating and/or preventing renal diseases. Renal diseases of particular interest include diabetic nephropathy, chronic kidney disease, proteinuria, and particularly acute kidney injury or acute renal failure (see Sharkovska et al. (2011) Clin. Lab. 57:507-515 and Newaz et al. (2010) Renal Failure 32:384-390). When used to treat renal disease, the compound may be administered in combination with other therapeutic agents such as angiotensin-converting enzyme inhibitors, AT₁ receptor antagonists, and diuretics.

Preventative Therapy

By potentiating the effects of the natriuretic peptides, compounds of the invention are also expected to be useful in preventative therapy, due to the antihypertrophic and antifibrotic effects of the natriuretic peptides (see Potter et al. (2009) Handbook of Experimental Pharmacology 191:341-366), for example in preventing the progression of cardiac insufficiency after myocardial infarction, preventing arterial restenosis after angioplasty, preventing thickening of blood vessel walls after vascular operations, preventing atherosclerosis, and preventing diabetic angiopathy.

Glaucoma

By potentiating the effects of the natriuretic peptides, compounds of the invention are expected to be useful to treat glaucoma. See, for example, Diestelhorst et al. (1989) International Ophthalmology 12:99-101. When used to treat glaucoma, compounds of the invention may be combined with one or more additional antiglaucoma agents.

Pain Relief

As NEP inhibitors, compounds of the invention are expected to inhibit the degradation of endogenous enkephalins and thus such compounds may also find utility as analgesics. See, for example, Rogues et al. (1980) Nature 288:286-288 and Thanawala et al. (2008) Current Drug Targets 9:887-894. When used to treat pain, the compounds of the invention may be combined with one or more additional antinociceptive drugs such as aminopeptidase N or dipeptidyl peptidase III inhibitors, non-steroidal anti-inflammatory agents, monoamine reuptake inhibitors, muscle relaxants, NMDA receptor antagonists, opioid receptor agonists, 5-HT_(1D) serotonin receptor agonists, and tricyclic antidepressants.

Other Utilities

Due to their NEP inhibition properties, compounds of the invention are also expected to be useful as antitussive agents, as well as find utility in the treatment of portal hypertension associated with liver cirrhosis (see Sansoe et al. (2005) J. Hepatol. 43:791-798), cancer (see Vesely (2005) J. Investigative Med. 53:360-365), depression (see Noble et al. (2007) Exp. Opin. Ther. Targets 11:145-159), menstrual disorders, preterm labor, pre-eclampsia, endometriosis, reproductive disorders (for example, male and female infertility, polycystic ovarian syndrome, implantation failure), and male and female sexual dysfunction, including male erectile dysfunction and female sexual arousal disorder. More specifically, the compounds of the invention are expected to be useful in treating female sexual dysfunction (see Pryde et al. (2006) J. Med. Chem. 49:4409-4424), which is often defined as a female patient's difficulty or inability to find satisfaction in sexual expression. This covers a variety of diverse female sexual disorders including, by way of illustration and not limitation, hypoactive sexual desire disorder, sexual arousal disorder, orgasmic disorder and sexual pain disorder. When used to treat such disorders, especially female sexual dysfunction, compounds of the invention may be combined with one or more of the following secondary agents: PDE-V inhibitors, dopamine agonists, estrogen receptor agonists and/or antagonists, androgens, and estrogens. Due to their NEP inhibition properties, compounds of the invention are also expected to have anti-inflammatory properties, and are expected to have utility as such, particularly when used in combination with statins.

Recent studies suggest that NEP plays a role in regulating nerve function in insulin-deficient diabetes and diet induced obesity. Coppey et al. (2011) Neuropharmacology 60:259-266. Therefore, due to their NEP inhibition properties, compounds of the invention are also expected to be useful in providing protection from nerve impairment caused by diabetes or diet induced obesity.

The amount of the compound of the invention administered per dose or the total amount administered per day may be predetermined or it may be determined on an individual patient basis by taking into consideration numerous factors, including the nature and severity of the patient's condition, the condition being treated, the age, weight, and general health of the patient, the tolerance of the patient to the active agent, the route of administration, pharmacological considerations such as the activity, efficacy, pharmacokinetics and toxicology profiles of the compound and any secondary agents being administered, and the like. Treatment of a patient suffering from a disease or medical condition (such as hypertension) can begin with a predetermined dosage or a dosage determined by the treating physician, and will continue for a period of time necessary to prevent, ameliorate, suppress, or alleviate the symptoms of the disease or medical condition. Patients undergoing such treatment will typically be monitored on a routine basis to determine the effectiveness of therapy. For example, in treating hypertension, blood pressure measurements may be used to determine the effectiveness of treatment. Similar indicators for other diseases and conditions described herein, are well known and are readily available to the treating physician. Continuous monitoring by the physician will insure that the optimal amount of the compound of the invention will be administered at any given time, as well as facilitating the determination of the duration of treatment. This is of particular value when secondary agents are also being administered, as their selection, dosage, and duration of therapy may also require adjustment. In this way, the treatment regimen and dosing schedule can be adjusted over the course of therapy so that the lowest amount of active agent that exhibits the desired effectiveness is administered and, further, that administration is continued only so long as is necessary to successfully treat the disease or medical condition.

Research Tools

Since compounds of the invention possess NEP enzyme inhibition activity, such compounds are also useful as research tools for investigating or studying biological systems or samples having a NEP enzyme, for example to study diseases where the NEP enzyme or its peptide substrates plays a role. Any suitable biological system or sample having a NEP enzyme may be employed in such studies which may be conducted either in vitro or in vivo. Representative biological systems or samples suitable for such studies include, but are not limited to, cells, cellular extracts, plasma membranes, tissue samples, isolated organs, mammals (such as mice, rats, guinea pigs, rabbits, dogs, pigs, humans, and so forth), and the like, with mammals being of particular interest. In one particular embodiment of the invention, NEP enzyme activity in a mammal is inhibited by administering a NEP-inhibiting amount of a compound of the invention. Compounds of the invention can also be used as research tools by conducting biological assays using such compounds.

When used as a research tool, a biological system or sample comprising a NEP enzyme is typically contacted with a NEP enzyme-inhibiting amount of a compound of the invention. After the biological system or sample is exposed to the compound, the effects of inhibiting the NEP enzyme are determined using conventional procedures and equipment, such as by measuring receptor binding in a binding assay or measuring ligand-mediated changes in a functional assay. Exposure encompasses contacting cells or tissue with the compound, administering the compound to a mammal, for example by i.p., p.o., i.v., s.c., or inhaled administration, and so forth. This determining step can involve measuring a response (a quantitative analysis) or can involve making an observation (a qualitative analysis). Measuring a response involves, for example, determining the effects of the compound on the biological system or sample using conventional procedures and equipment, such as enzyme activity assays and measuring enzyme substrate or product mediated changes in functional assays. The assay results can be used to determine the activity level as well as the amount of compound necessary to achieve the desired result, that is, a NEP enzyme-inhibiting amount. Typically, the determining step will involve determining the effects of inhibiting the NEP enzyme.

Additionally, compounds of the invention can be used as research tools for evaluating other chemical compounds, and thus are also useful in screening assays to discover, for example, new compounds having NEP-inhibiting activity. In this manner, a compound of the invention is used as a standard in an assay to allow comparison of the results obtained with a test compound and with compounds of the invention to identify those test compounds that have about equal or superior activity, if any. For example, pK_(i), data for a test compound or a group of test compounds is compared to the pK_(i) data for a compound of the invention to identify those test compounds that have the desired properties, for example, test compounds having a pK_(i) value about equal or superior to a compound of the invention, if any. This aspect of the invention includes, as separate embodiments, both the generation of comparison data (using the appropriate assays) and the analysis of test data to identify test compounds of interest. Thus, a test compound can be evaluated in a biological assay, by a method comprising the steps of: (a) conducting a biological assay with a test compound to provide a first assay value; (b) conducting the biological assay with a compound of the invention to provide a second assay value; wherein step (a) is conducted either before, after or concurrently with step (b); and (c) comparing the first assay value from step (a) with the second assay value from step (b). Exemplary biological assays include a NEP enzyme inhibition assay.

Pharmaceutical Compositions and Formulations

Compounds of the invention are typically administered to a patient in the form of a pharmaceutical composition or formulation. Such pharmaceutical compositions may be administered to the patient by any acceptable route of administration including, but not limited to, oral, rectal, vaginal, nasal, inhaled, topical (including transdermal), ocular, and parenteral modes of administration. Further, the compounds of the invention may be administered, for example orally, in multiple doses per day (for example, two, three, or four times daily), in a single daily dose or a single weekly dose. It will be understood that any form of the compounds of the invention, (that is, free base, free acid, pharmaceutically acceptable salt, solvate, etc.) that is suitable for the particular mode of administration can be used in the pharmaceutical compositions discussed herein.

Accordingly, in one embodiment, the invention relates to a pharmaceutical composition comprising a pharmaceutically acceptable carrier and a compound of the invention. The compositions may contain other therapeutic and/or formulating agents if desired. When discussing compositions, the “compound of the invention” may also be referred to herein as the “active agent,” to distinguish it from other components of the formulation, such as the carrier. Thus, it is understood that the term “active agent” includes compounds of formula I as well as pharmaceutically acceptable salts, solvates and prodrugs of that compound.

The pharmaceutical compositions of the invention typically contain a therapeutically effective amount of a compound of the invention. Those skilled in the art will recognize, however, that a pharmaceutical composition may contain more than a therapeutically effective amount, such as in bulk compositions, or less than a therapeutically effective amount, that is, individual unit doses designed for multiple administration to achieve a therapeutically effective amount. Typically, the composition will contain from about 0.01-95 wt % of active agent, including, from about 0.01-30 wt %, such as from about 0.01-10 wt %, with the actual amount depending upon the formulation itself, the route of administration, the frequency of dosing, and so forth. In one embodiment, a composition suitable for an oral dosage form, for example, may contain about 5-70 wt %, or from about 10-60 wt % of active agent.

Any conventional carrier or excipient may be used in the pharmaceutical compositions of the invention. The choice of a particular carrier or excipient, or combinations of carriers or excipients, will depend on the mode of administration being used to treat a particular patient or type of medical condition or disease state. In this regard, the preparation of a suitable composition for a particular mode of administration is well within the scope of those skilled in the pharmaceutical arts. Additionally, carriers or excipients used in such compositions are commercially available. By way of further illustration, conventional formulation techniques are described in Remington: The Science and Practice of Pharmacy, 20^(th) Edition, Lippincott Williams & White, Baltimore, Md. (2000); and H. C. Ansel et al., Pharmaceutical Dosage Forms and Drug Delivery Systems, 7^(th) Edition, Lippincott Williams & White, Baltimore, Md. (1999).

Representative examples of materials which can serve as pharmaceutically acceptable carriers include, but are not limited to, the following: sugars, such as lactose, glucose and sucrose; starches, such as corn starch and potato starch; cellulose, such as microcrystalline cellulose, and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; powdered tragacanth; malt; gelatin; talc; excipients, such as cocoa butter and suppository waxes; oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; glycols, such as propylene glycol; polyols, such as glycerin, sorbitol, mannitol and polyethylene glycol; esters, such as ethyl oleate and ethyl laurate; agar; buffering agents, such as magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-free water; isotonic saline; Ringer's solution; ethyl alcohol; phosphate buffer solutions; compressed propellant gases, such as chlorofluorocarbons and hydrofluorocarbons; and other non-toxic compatible substances employed in pharmaceutical compositions.

Pharmaceutical compositions are typically prepared by thoroughly and intimately mixing or blending the active agent with a pharmaceutically acceptable carrier and one or more optional ingredients. The resulting uniformly blended mixture may then be shaped or loaded into tablets, capsules, pills, canisters, cartridges, dispensers and the like using conventional procedures and equipment.

In one embodiment, the pharmaceutical compositions are suitable for oral administration. Suitable compositions for oral administration may be in the form of capsules, tablets, pills, lozenges, cachets, dragees, powders, granules; solutions or suspensions in an aqueous or non-aqueous liquid; oil-in-water or water-in-oil liquid emulsions; elixirs or syrups; and the like; each containing a predetermined amount of the active agent.

When intended for oral administration in a solid dosage form (capsules, tablets, pills and the like), the composition will typically comprise the active agent and one or more pharmaceutically acceptable carriers, such as sodium citrate or dicalcium phosphate. Solid dosage forms may also comprise: fillers or extenders, such as starches, microcrystalline cellulose, lactose, sucrose, glucose, mannitol, and/or silicic acid; binders, such as carboxymethylcellulose, alginates, gelatin, polyvinyl pyrrolidone, sucrose and/or acacia; humectants, such as glycerol; disintegrating agents, such as agar-agar, calcium carbonate, potato or tapioca starch, alginic acid, certain silicates, and/or sodium carbonate; solution retarding agents, such as paraffin; absorption accelerators, such as quaternary ammonium compounds; wetting agents, such as cetyl alcohol and/or glycerol monostearate; absorbents, such as kaolin and/or bentonite clay; lubricants, such as talc, calcium stearate, magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate, and/or mixtures thereof; coloring agents; and buffering agents.

Release agents, wetting agents, coating agents, sweetening, flavoring and perfuming agents, preservatives and antioxidants may also be present in the pharmaceutical compositions. Exemplary coating agents for tablets, capsules, pills and like, include those used for enteric coatings, such as cellulose acetate phthalate, polyvinyl acetate phthalate, hydroxypropyl methylcellulose phthalate, methacrylic acid-methacrylic acid ester copolymers, cellulose acetate trimellitate, carboxymethyl ethyl cellulose, hydroxypropyl methyl cellulose acetate succinate, and the like. Examples of pharmaceutically acceptable antioxidants include: water-soluble antioxidants, such as ascorbic acid, cysteine hydrochloride, sodium bisulfate, sodium metabisulfate, sodium sulfite and the like; oil-soluble antioxidants, such as ascorbyl palmitate, butylated hydroxyanisole, butylated hydroxytoluene, lecithin, propyl gallate, alpha-tocopherol, and the like; and metal-chelating agents, such as citric acid, ethylenediamine tetraacetic acid, sorbitol, tartaric acid, phosphoric acid, and the like.

Compositions may also be formulated to provide slow or controlled release of the active agent using, by way of example, hydroxypropyl methyl cellulose in varying proportions or other polymer matrices, liposomes and/or microspheres. In addition, the pharmaceutical compositions of the invention may contain opacifying agents and may be formulated so that they release the active agent only, or preferentially, in a certain portion of the gastrointestinal tract, optionally, in a delayed manner. Examples of embedding compositions which can be used include polymeric substances and waxes. The active agent can also be in micro-encapsulated form, optionally with one or more of the above-described excipients.

Suitable liquid dosage forms for oral administration include, by way of illustration, pharmaceutically acceptable emulsions, microemulsions, solutions, suspensions, syrups and elixirs. Liquid dosage forms typically comprise the active agent and an inert diluent, such as, for example, water or other solvents, solubilizing agents and emulsifiers, such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, oils (for example, cottonseed, groundnut, corn, germ, olive, castor and sesame oils), glycerol, tetrahydrofuryl alcohol, polyethylene glycols and fatty acid esters of sorbitan, and mixtures thereof. Suspensions may contain suspending agents such as, for example, ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminium metahydroxide, bentonite, agar-agar and tragacanth, and mixtures thereof.

When intended for oral administration, the pharmaceutical compositions of the invention may be packaged in a unit dosage form. The term “unit dosage form” refers to a physically discrete unit suitable for dosing a patient, that is, each unit containing a predetermined quantity of the active agent calculated to produce the desired therapeutic effect either alone or in combination with one or more additional units. For example, such unit dosage forms may be capsules, tablets, pills, and the like.

In another embodiment, the compositions of the invention are suitable for inhaled administration, and will typically be in the form of an aerosol or a powder. Such compositions are generally administered using well-known delivery devices, such as a nebulizer, dry powder, or metered-dose inhaler. Nebulizer devices produce a stream of high velocity air that causes the composition to spray as a mist that is carried into a patient's respiratory tract. An exemplary nebulizer formulation comprises the active agent dissolved in a carrier to form a solution, or micronized and combined with a carrier to form a suspension of micronized particles of respirable size. Dry powder inhalers administer the active agent as a free-flowing powder that is dispersed in a patient's air-stream during inspiration. An exemplary dry powder formulation comprises the active agent dry-blended with an excipient such as lactose, starch, mannitol, dextrose, polylactic acid, polylactide-co-glycolide, and combinations thereof. Metered-dose inhalers discharge a measured amount of the active agent using compressed propellant gas. An exemplary metered-dose formulation comprises a solution or suspension of the active agent in a liquefied propellant, such as a chlorofluorocarbon or hydrofluoroalkane. Optional components of such formulations include co-solvents, such as ethanol or pentane, and surfactants, such as sorbitan trioleate, oleic acid, lecithin, glycerin, and sodium lauryl sulfate. Such compositions are typically prepared by adding chilled or pressurized hydrofluoroalkane to a suitable container containing the active agent, ethanol (if present) and the surfactant (if present). To prepare a suspension, the active agent is micronized and then combined with the propellant. Alternatively, a suspension formulation can be prepared by spray drying a coating of surfactant on micronized particles of the active agent. The formulation is then loaded into an aerosol canister, which forms a portion of the inhaler.

Compounds of the invention can also be administered parenterally (for example, by subcutaneous, intravenous, intramuscular, or intraperitoneal injection). For such administration, the active agent is provided in a sterile solution, suspension, or emulsion. Exemplary solvents for preparing such formulations include water, saline, low molecular weight alcohols such as propylene glycol, polyethylene glycol, oils, gelatin, fatty acid esters such as ethyl oleate, and the like. Parenteral formulations may also contain one or more anti-oxidants, solubilizers, stabilizers, preservatives, wetting agents, emulsifiers, and dispersing agents. Surfactants, additional stabilizing agents or pH-adjusting agents (acids, bases or buffers) and anti-oxidants are particularly useful to provide stability to the formulation, for example, to minimize or avoid hydrolysis of ester and amide linkages that may be present in the compound. These formulations may be rendered sterile by use of a sterile injectable medium, a sterilizing agent, filtration, irradiation, or heat. In one particular embodiment, the parenteral formulation comprises an aqueous cyclodextrin solution as the pharmaceutically acceptable carrier. Suitable cyclodextrins include cyclic molecules containing six or more α-D-glucopyranose units linked at the 1,4 positions by a linkages as in amylase, β-cyclodextrin or cycloheptaamylose. Exemplary cyclodextrins include cyclodextrin derivatives such as hydroxypropyl and sulfobutyl ether cyclodextrins such as hydroxypropyl-β-cyclodextrin and sulfobutyl ether β-cyclodextrin. Exemplary buffers for such formulations include carboxylic acid-based buffers such as citrate, lactate and maleate buffer solutions.

Compounds of the invention can also be administered transdermally using known transdermal delivery systems and excipients. For example, the compound can be admixed with permeation enhancers, such as propylene glycol, polyethylene glycol monolaurate, azacycloalkan-2-ones and the like, and incorporated into a patch or similar delivery system. Additional excipients including gelling agents, emulsifiers and buffers, may be used in such transdermal compositions if desired.

Secondary Agents

The compounds of the invention may be useful as the sole treatment of a disease or may be combined with one or more additional therapeutic agents in order to obtain the desired therapeutic effect. Thus, in one embodiment, pharmaceutical compositions of the invention contain other drugs that are co-administered with a compound of the invention. For example, the composition may further comprise one or more drugs (also referred to as “secondary agents(s)”). Such therapeutic agents are well known in the art, and include adenosine receptor antagonists, α-adrenergic receptor antagonists, β₁-adrenergic receptor antagonists, β₂-adrenergic receptor agonists, dual-acting β-adrenergic receptor antagonist/α₁-receptor antagonists, advanced glycation end product breakers, aldosterone antagonists, aldosterone synthase inhibitors, aminopeptidase N inhibitors, androgens, angiotensin-converting enzyme inhibitors and dual-acting angiotensin-converting enzyme/neprilysin inhibitors, angiotensin-converting enzyme 2 activators and stimulators, angiotensin-II vaccines, anticoagulants, anti-diabetic agents, antidiarrheal agents, anti-glaucoma agents, anti-lipid agents, antinociceptive agents, anti-thrombotic agents, AT₁ receptor antagonists and dual-acting AT₁ receptor antagonist/neprilysin inhibitors and multifunctional angiotensin receptor blockers, bradykinin receptor antagonists, calcium channel blockers, chymase inhibitors, digoxin, diuretics, dopamine agonists, endothelin converting enzyme inhibitors, endothelin receptor antagonists, HMG-CoA reductase inhibitors, estrogens, estrogen receptor agonists and/or antagonists, monoamine reuptake inhibitors, muscle relaxants, natriuretic peptides and their analogs, natriuretic peptide clearance receptor antagonists, neprilysin inhibitors, nitric oxide donors, non-steroidal anti-inflammatory agents, N-methyl d-aspartate receptor antagonists, opioid receptor agonists, phosphodiesterase inhibitors, prostaglandin analogs, prostaglandin receptor agonists, renin inhibitors, selective serotonin reuptake inhibitors, sodium channel blocker, soluble guanylate cyclase stimulators and activators, tricyclic antidepressants, vasopressin receptor antagonists, and combinations thereof. Specific examples of these agents are detailed herein.

Accordingly, in yet another aspect of the invention, a pharmaceutical composition comprises a compound of the invention, a second active agent, and a pharmaceutically acceptable carrier. Third, fourth etc. active agents may also be included in the composition. In combination therapy, the amount of compound of the invention that is administered, as well as the amount of secondary agents, may be less than the amount typically administered in monotherapy.

Compounds of the invention may be physically mixed with the second active agent to form a composition containing both agents; or each agent may be present in separate and distinct compositions which are administered to the patient simultaneously or at separate times. For example, a compound of the invention can be combined with a second active agent using conventional procedures and equipment to form a combination of active agents comprising a compound of the invention and a second active agent. Additionally, the active agents may be combined with a pharmaceutically acceptable carrier to form a pharmaceutical composition comprising a compound of the invention, a second active agent and a pharmaceutically acceptable carrier. In this embodiment, the components of the composition are typically mixed or blended to create a physical mixture. The physical mixture is then administered in a therapeutically effective amount using any of the routes described herein.

Alternatively, the active agents may remain separate and distinct before administration to the patient. In this embodiment, the agents are not physically mixed together before administration but are administered simultaneously or at separate times as separate compositions. Such compositions can be packaged separately or may be packaged together in a kit. When administered at separate times, the secondary agent will typically be administered less than 24 hours after administration of the compound of the invention, ranging anywhere from concurrent with administration of the compound of the invention to about 24 hours post-dose. This is also referred to as sequential administration. Thus, a compound of the invention can be orally administered simultaneously or sequentially with another active agent using two tablets, with one tablet for each active agent, where sequential may mean being administered immediately after administration of the compound of the invention or at some predetermined time later (for example, one hour later or three hours later). It is also contemplated that the secondary agent may be administered more than 24 hours after administration of the compound of the invention. Alternatively, the combination may be administered by different routes of administration, that is, one orally and the other by inhalation.

In one embodiment, the kit comprises a first dosage form comprising a compound of the invention and at least one additional dosage form comprising one or more of the secondary agents set forth herein, in quantities sufficient to carry out the methods of the invention. The first dosage form and the second (or third, etc.) dosage form together comprise a therapeutically effective amount of active agents for the treatment or prevention of a disease or medical condition in a patient.

Secondary agent(s), when included, are present in a therapeutically effective amount such that they are typically administered in an amount that produces a therapeutically beneficial effect when co-administered with a compound of the invention. The secondary agent can be in the form of a pharmaceutically acceptable salt, solvate, optically pure stereoisomer, and so forth. The secondary agent may also be in the form of a prodrug, for example, a compound having a carboxylic acid group that has been esterified. Thus, secondary agents listed herein are intended to include all such forms, and are commercially available or can be prepared using conventional procedures and reagents.

In one embodiment, compounds of the invention are administered in combination with an adenosine receptor antagonist, representative examples of which include, but are not limited to, naxifylline, rolofylline, SLV-320, theophylline, and tonapofylline.

In one embodiment, compounds of the invention are administered in combination with an α-adrenergic receptor antagonist, representative examples of which include, but are not limited to, doxazosin, prazosin, tamsulosin, and terazosin.

Compounds of the invention may also be administered in combination with a β₁-adrenergic receptor antagonist (“β₁-blockers”). Representative β₁-blockers include, but are not limited to, acebutolol, alprenolol, amosulalol, arotinolol, atenolol, befunolol, betaxolol, bevantolol, bisoprolol, bopindolol, bucindolol, bucumolol, bufetolol, bufuralol, bunitrolol, bupranolol, bubridine, butofilolol, carazolol, carteolol, carvedilol, celiprolol, cetamolol, cloranolol, dilevalol, epanolol, esmolol, indenolol, labetolol, levobunolol, mepindolol, metipranolol, metoprolol such as metoprolol succinate and metoprolol tartrate, moprolol, nadolol, nadoxolol, nebivalol, nipradilol, oxprenolol, penbutolol, perbutolol, pindolol, practolol, pronethalol, propranolol, sotalol, sufinalol, talindol, tertatolol, tilisolol, timolol, toliprolol, xibenolol, and combinations thereof. In one particular embodiment, the β₁-antagonist is selected from atenolol, bisoprolol, metoprolol, propranolol, sotalol, and combinations thereof. Typically, the β₁-blocker will be administered in an amount sufficient to provide from about 2-900 mg per dose.

In one embodiment, compounds of the invention are administered in combination with a β₂-adrenergic receptor agonist, representative examples of which include, but are not limited to, albuterol, bitolterol, fenoterol, formoterol, indacaterol, isoetharine, levalbuterol, metaproterenol, pirbuterol, salbutamol, salmefamol, salmeterol, terbutaline, vilanterol, and the like. Typically, the β₂-adrenoreceptor agonist will be administered in an amount sufficient to provide from about 0.05-500 μg per dose.

In one embodiment, compounds of the invention are administered in combination with an advanced glycation end product (AGE) breaker, examples of which include, by way of illustration and not limitation, alagebrium (or ALT-711), and TRC4149.

In another embodiment, compounds of the invention are administered in combination with an aldosterone antagonist, representative examples of which include, but are not limited to, eplerenone, spironolactone, and combinations thereof. Typically, the aldosterone antagonist will be administered in an amount sufficient to provide from about 5-300 mg per day.

In one embodiment, compounds of the invention are administered in combination with an aminopeptidase N or dipeptidyl peptidase III inhibitor, examples of which include, by way of illustration and not limitation, bestatin and PC18 (2-amino-4-methylsulfonyl butane thiol, methionine thiol).

Compounds of the invention can also be administered in combination with an angiotensin-converting enzyme (ACE) inhibitor. Representative ACE inhibitors include, but are not limited to, accupril, alacepril, benazepril, benazeprilat, captopril, ceranapril, cilazapril, delapril, enalapril, enalaprilat, fosinopril, fosinoprilat, imidapril, lisinopril, moexipril, monopril, moveltipril, pentopril, perindopril, quinapril, quinaprilat, ramipril, ramiprilat, saralasin acetate, spirapril, temocapril, trandolapril, zofenopril, and combinations thereof.

In a particular embodiment, the ACE inhibitor is selected from: benazepril, captopril, enalapril, lisinopril, ramipril, and combinations thereof. Typically, the ACE inhibitor will be administered in an amount sufficient to provide from about 1-150 mg per day. In another embodiment, compounds of the invention are administered in combination with a dual-acting angiotensin-converting enzyme/neprilysin (ACE/NEP) inhibitor, examples of which include, but are not limited to: AVE-0848 ((4S,7S,12bR)-7-[3-methyl-2(S)-sulfanylbutyramido]-6-oxo-1,2,3,4,6,7,8,12b-octahydropyrido[2,1-a][2]-benzazepine-4-carboxylic acid); AVE-7688 (ilepatril) and its parent compound; BMS-182657 (2-[2-oxo-3(S)-[3-phenyl-2(S)-sulfanylpropionamido]-2,3,4,5-tetrahydro-1H-1-benzazepin-1-yl]acetic acid); CGS-35601 (N-[1-[4-methyl-2(S)-sulfanylpentanamido]cyclopentylcarbonyl]-L-tryptophan); fasidotril; fasidotrilate; enalaprilat; ER-32935 ((3R,6S,9aR)-6-[3(S)-methyl-2(S)-sulfanylpentanamido]-5-oxoperhydrothiazolo[3,2-a]azepine-3-carboxylic acid); gempatrilat; MDL-101264 ((4S,7S,12bR)-7-[2(S)-(2-morpholinoacetylthio)-3-phenylpropionamido]-6-oxo-1,2,3,4,6,7,8,12b-octahydropyrido[2,1-a][2]benzazepine-4-carboxylic acid); MDL-101287 ([4S-[4α,7α(R*),12bβ]]-7-[2-(carboxymethyl)-3-phenylpropionamido]-6-oxo-1,2,3,4,6,7,8,12b-octahydropyrido[2,1-a][2]benzazepine-4-carboxylic acid); omapatrilat; RB-105 (N-[2(S)-(mercaptomethyl)-3(R)-phenylbutyl]-L-alanine); sampatrilat; SA-898 ((2R,4R)-N-[2-(2-hydroxyphenyl)-3-(3-mercaptopropionyl)thiazolidin-4-ylcarbonyl]-L-phenylalanine); Sch-50690 (N-[1(S)-carboxy-2-[N2-(methanesulfonyl)-L-lysylamino]ethyl]-L-valyl-L-tyrosine); and combinations thereof, may also be included. In one particular embodiment, the ACE/NEP inhibitor is selected from: AVE-7688, enalaprilat, fasidotril, fasidotrilate, omapatrilat, sampatrilat, and combinations thereof.

In one embodiment, compounds of the invention are administered in combination with an angiotensin-converting enzyme 2 (ACE2) activator or stimulator.

In one embodiment, compounds of the invention are administered in combination with an angiotensin-II vaccine, examples of which include, but are not limited to ATR12181 and CYT006-AngQb.

In one embodiment, compounds of the invention are administered in combination with an anticoagulant, representative examples of which include, but are not limited to: coumarins such as warfarin; heparin; and direct thrombin inhibitors such as argatroban, bivalirudin, dabigatran, and lepirudin.

In yet another embodiment, compounds of the invention are administered in combination with an anti-diabetic agent. Representative anti-diabetic agents include injectable drugs as well as orally effective drugs, and combinations thereof. Examples of injectable drugs include, but are not limited to, insulin and insulin derivatives. Examples of orally effective drugs include, but are not limited to: biguanides such as metformin; glucagon antagonists; α-glucosidase inhibitors such as acarbose and miglitol; dipeptidyl peptidase IV inhibitors (DPP-IV inhibitors) such as alogliptin, denagliptin, linagliptin, saxagliptin, sitagliptin, and vildagliptin; meglitinides such as repaglinide; oxadiazolidinediones; sulfonylureas such as chlorpropamide, glimepiride, glipizide, glyburide, and tolazamide; thiazolidinediones such as pioglitazone and rosiglitazone; and combinations thereof.

In another embodiment, compounds of the invention are administered in combination with antidiarrheal treatments. Representative treatment options include, but are not limited to, oral rehydration solutions (ORS), loperamide, diphenoxylate, and bismuth subsalicylate.

In yet another embodiment, a compound of the invention is administered in combination with an anti-glaucoma agent. Representative anti-glaucoma agents include, but are not limited to: α-adrenergic agonists such as brimonidine; β₁-adrenergic receptor antagonists; topical β₁-blockers such as betaxolol, levobunolol, and timolol; carbonic anhydrase inhibitors such as acetazolamide, brinzolamide, or dorzolamide; cholinergic agonists such as cevimeline and DMXB-anabaseine; epinephrine compounds; miotics such as pilocarpine; and prostaglandin analogs.

In yet another embodiment, compounds of the invention are administered in combination with an anti-lipid agent. Representative anti-lipid agents include, but are not limited to: cholesteryl ester transfer protein inhibitors (CETPs) such as anacetrapib, dalcetrapib, and torcetrapib; statins such as atorvastatin, fluvastatin, lovastatin, pravastatin, rosuvastatin and simvastatin; and combinations thereof.

In one embodiment, compounds of the invention are administered in combination with an anti-thrombotic agent. Representative anti-thrombotic agents include, but are not limited to: aspirin; anti-platelet agents such as clopidogrel, prasugrel, and ticlopidine; heparin, and combinations thereof.

In one embodiment, compounds of the invention are administered in combination with an AT₁ receptor antagonist, also known as angiotensin II type 1 receptor blockers (ARBs). Representative ARBs include, but are not limited to, abitesartan, azilsartan (e.g., azilsartan medoxomil), benzyllosartan, candesartan, candesartan cilexetil, elisartan, embusartan, enoltasosartan, eprosartan, EXP3174, fonsartan, forasartan, glycyllosartan, irbesartan, isoteoline, losartan, milfasartan, olmesartan (e.g., olmesartan medoxomil), opomisartan, pratosartan, ripisartan, saprisartan, saralasin, sarmesin, TAK-591, tasosartan, telmisartan, valsartan, zolasartan, and combinations thereof. In a particular embodiment, the ARB is selected from azilsartan medoxomil, candesartan cilexetil, eprosartan, irbesartan, losartan, olmesartan medoxomil, saprisartan, tasosartan, telmisartan, valsartan, and combinations thereof. Exemplary salts and/or prodrugs include candesartan cilexetil, eprosartan mesylate, losartan potassium salt, and olmesartan medoxomil. Typically, the ARB will be administered in an amount sufficient to provide from about 4-600 mg per dose, with exemplary daily dosages ranging from 20-320 mg per day.

Compounds of the invention may also be administered in combination with a dual-acting agent, such as an AT₁ receptor antagonist/neprilysin inhibitor (ARB/NEP) inhibitor, examples of which include, but are not limited to, compounds described in U.S. Publication Nos. 2008/0269305 and 2009/0023228, both to Allegretti et al. filed on Apr. 23, 2008, such as the compound, 4′-{2-ethoxy-4-ethyl-5-[((S)-2-mercapto-4-methylpentanoylamino)-methyl]imidazol-1-ylmethyl}-3′-fluorobiphenyl-2-carboxylic acid.

Compounds of the invention may also be administered in combination with multifunctional angiotensin receptor blockers as described in Kurtz & Klein (2009) Hypertension Research 32:826-834.

In one embodiment, compounds of the invention are administered in combination with a bradykinin receptor antagonist, for example, icatibant (HOE-140). It is expected that this combination therapy may present the advantage of preventing angioedema or other unwanted consequences of elevated bradykinin levels.

In one embodiment, compounds of the invention are administered in combination with a calcium channel blocker. Representative calcium channel blockers include, but are not limited to, amlodipine, anipamil, aranipine, barnidipine, bencyclane, benidipine, bepridil, clentiazem, cilnidipine, cinnarizine, diltiazem, efonidipine, elgodipine, etafenone, felodipine, fendiline, flunarizine, gallopamil, isradipine, lacidipine, lercanidipine, lidoflazine, lomerizine, manidipine, mibefradil, nicardipine, nifedipine, niguldipine, niludipine, nilvadipine, nimodipine, nisoldipine, nitrendipine, nivaldipine, perhexyline, prenylamine, ryosidine, semotiadil, terodiline, tiapamil, verapamil, and combinations thereof. In a particular embodiment, the calcium channel blocker is selected from amlodipine, bepridil, diltiazem, felodipine, isradipine, lacidipine, nicardipine, nifedipine, niguldipine, niludipine, nimodipine, nisoldipine, ryosidine, verapamil, and combinations thereof. Typically, the calcium channel blocker will be administered in an amount sufficient to provide from about 2-500 mg per dose.

In one embodiment, compounds of the invention are administered in combination with a chymase inhibitor, such as TPC-806 and 2-(5-formylamino-6-oxo-2-phenyl-1,6-dihydropyrimidine-1-yl)-N-[{3,4-dioxo-1-phenyl-7-(2-pyridyloxy)}-2-heptyl]acetamide (NK3201).

In one embodiment, compounds of the invention are administered in combination with a diuretic. Representative diuretics include, but are not limited to: carbonic anhydrase inhibitors such as acetazolamide and dichlorphenamide; loop diuretics, which include sulfonamide derivatives such as acetazolamide, ambuside, azosemide, bumetanide, butazolamide, chloraminophenamide, clofenamide, clopamide, clorexolone, disulfamide, ethoxzolamide, furosemide, mefruside, methazolamide, piretanide, torsemide, tripamide, and xipamide, as well as non-sulfonamide diuretics such as ethacrynic acid and other phenoxyacetic acid compounds such as tienilic acid, indacrinone and quincarbate; osmotic diuretics such as mannitol; potassium-sparing diuretics, which include aldosterone antagonists such as spironolactone, and Na⁺ channel inhibitors such as amiloride and triamterene; thiazide and thiazide-like diuretics such as althiazide, bendroflumethiazide, benzylhydrochlorothiazide, benzthiazide, buthiazide, chlorthalidone, chlorothiazide, cyclopenthiazide, cyclothiazide, epithiazide, ethiazide, fenquizone, flumethiazide, hydrochlorothiazide, hydroflumethiazide, indapamide, methylclothiazide, meticrane, metolazone, paraflutizide, polythiazide, quinethazone, teclothiazide, and trichloromethiazide; and combinations thereof. In a particular embodiment, the diuretic is selected from amiloride, bumetanide, chlorothiazide, chlorthalidone, dichlorphenamide, ethacrynic acid, furosemide, hydrochlorothiazide, hydroflumethiazide, indapamide, methylclothiazide, metolazone, torsemide, triamterene, and combinations thereof. The diuretic will be administered in an amount sufficient to provide from about 5-50 mg per day, more typically 6-25 mg per day, with common dosages being 6.25 mg, 12.5 mg or 25 mg per day.

Compounds of the invention may also be administered in combination with an endothelin converting enzyme (ECE) inhibitor, examples of which include, but are not limited to, phosphoramidon, CGS 26303, and combinations thereof.

In a particular embodiment, compounds of the invention are administered in combination with an endothelin receptor antagonist. Representative endothelin receptor antagonists include, but are not limited to: selective endothelin receptor antagonists that affect endothelin A receptors, such as avosentan, ambrisentan, atrasentan, BQ-123, clazosentan, darusentan, sitaxentan, and zibotentan; and dual endothelin receptor antagonists that affect both endothelin A and B receptors, such as bosentan, macitentan, tezosentan).

In yet another embodiment, a compound of the invention is administered in combination with one or more HMG-CoA reductase inhibitors, which are also known as statins. Representative statins include, but are not limited to, atorvastatin, fluvastatin, lovastatin, pitavastatin, pravastatin, rosuvastatin and simvastatin.

In one embodiment, compounds of the invention are administered in combination with a monoamine reuptake inhibitor, examples of which include, by way of illustration and not limitation, norepinephrine reuptake inhibitors such as atomoxetine, buproprion and the buproprion metabolite hydroxybuproprion, maprotiline, reboxetine, and viloxazine; selective serotonin reuptake inhibitors (SSRIs) such as citalopram and the citalopram metabolite desmethylcitalopram, dapoxetine, escitalopram (e.g., escitalopram oxalate), fluoxetine and the fluoxetine desmethyl metabolite norfluoxetine, fluvoxamine (e.g., fluvoxamine maleate), paroxetine, sertraline and the sertraline metabolite demethylsertraline; dual serotonin-norepinephrine reuptake inhibitors (SNRIs) such as bicifadine, duloxetine, milnacipran, nefazodone, and venlafaxine; and combinations thereof.

In another embodiment, compounds of the invention are administered in combination with a muscle relaxant, examples of which include, but are not limited to: carisoprodol, chlorzoxazone, cyclobenzaprine, diflunisal, metaxalone, methocarbamol, and combinations thereof.

In one embodiment, compounds of the invention are administered in combination with a natriuretic peptide or analog, examples of which include but are not limited to: carperitide, CD-NP (Nile Therapeutics), CU-NP, nesiritide, PL-3994 (Palatin Technologies, Inc.), ularitide, cenderitide, and compounds described in Ogawa et al (2004) J. Biol. Chem. 279:28625-31. These compounds are also referred to as natriuretic peptide receptor-A (NPR-A) agonists. In another embodiment, compounds of the invention are administered in combination with a natriuretic peptide clearance receptor (NPR-C) antagonist such as SC-46542, cANF (4-23), and AP-811 (Veale (2000) Bioorg Med Chem Lett 10:1949-52). For example, AP-811 has shown synergy when combined with the NEP inhibitor, thiorphan (Wegner (1995) Clin. Exper. Hypert. 17:861-876).

In another embodiment, compounds of the invention are administered in combination with a neprilysin (NEP) inhibitor. Representative NEP inhibitors include, but are not limited to: AHU-377; candoxatril; candoxatrilat; dexecadotril ((+)-N-[2(R)-(acetylthiomethyl)-3-phenylpropionyl]glycine benzyl ester); CGS-24128 (3-[3-(biphenyl-4-yl)-2-(phosphonomethylamino)propionamido]propionic acid); CGS-24592 ((S)-3-[3-(biphenyl-4-yl)-2-(phosphonomethylamino)propionamido]propionic acid); CGS-25155 (N-[9(R)-(acetylthiomethyl)-10-oxo-1-azacyclodecan-2(S)-ylcarbonyl]-4(R)-hydroxy-L-proline benzyl ester); 3-(1-carbamoylcyclohexyl)propionic acid derivatives described in WO 2006/027680 to Hepworth et al. (Pfizer Inc.); JMV-390-1 (2(R)-benzyl-3-(N-hydroxycarbamoyl)propionyl-L-isoleucyl-L-leucine); ecadotril; phosphoramidon; retrothiorphan; RU-42827 (2-(mercaptomethyl)-N-(4-pyridinyl)benzenepropionamide); RU-44004 (N-(4-morpholinyl)-3-phenyl-2-(sulfanylmethyl)propionamide); SCH-32615 ((S)-N—[N-(1-carboxy-2-phenylethyl)-L-phenylalanyl]-β-alanine) and its prodrug SCH-34826 ((S)-N—[N-[1-[[(2,2-dimethyl-1,3-dioxolan-4-yl)methoxy]carbonyl]-2-phenylethyl]-L-phenylalanyl]-β-alanine); sialorphin; SCH-42495 (N-[2(S)-(acetylsulfanylmethyl)-3-(2-methylphenyl)propionyl]-L-methionine ethyl ester); spinorphin; SQ-28132 (N-[2-(mercaptomethyl)-1-oxo-3-phenylpropyl]leucine); SQ-28603 (N-[2-(mercaptomethyl)-1-oxo-3-phenylpropyl]-β-alanine); SQ-29072 (7-[[2-(mercaptomethyl)-1-oxo-3-phenylpropyl]amino]heptanoic acid); thiorphan and its prodrug racecadotril; UK-69578 (cis-4-[[[1-[2-carboxy-3-(2-methoxyethoxy)propyl]cyclopentyl]carbonyl]amino]cyclohexanecarboxylic acid); UK-447,841 (2-{1-[3-(4-chlorophenyl)propylcarbamoyl]-cyclopentylmethyl}-4-methoxybutyric acid); UK-505,749 ((R)-2-methyl-3-{1-[3-(2-methylbenzothiazol-6-yl)propylcarbamoyl]cyclopentyl}propionic acid); 5-biphenyl-4-yl-4-(3-carboxypropionylamino)-2-methylpentanoic acid and 5-biphenyl-4-yl-4-(3-carboxypropionylamino)-2-methylpentanoic acid ethyl ester (WO 2007/056546); daglutril [(3S,2′R)-3-{1-[2′-(ethoxycarbonyl)-4′-phenylbutyl]-cyclopentan-1-carbonylamino}-2,3,4,5-tetrahydro-2-oxo-1H-1-benzazepine-1-acetic acid] described in WO 2007/106708 to Khder et al. (Novartis AG); and combinations thereof. In a particular embodiment, the NEP inhibitor is selected from AHU-377, candoxatril, candoxatrilat, CGS-24128, phosphoramidon, SCH-32615, SCH-34826, SQ-28603, thiorphan, and combinations thereof. In a particular embodiment, the NEP inhibitor is a compound such as daglutril or CGS-26303 ([N-[2-(biphenyl-4-yl)-1(S)-(1H-tetrazol-5-yl)ethyl]amino]methylphosphonic acid), which have activity both as inhibitors of the endothelin converting enzyme (ECE) and of NEP. Other dual acting ECE/NEP compounds can also be used. The NEP inhibitor will be administered in an amount sufficient to provide from about 20-800 mg per day, with typical daily dosages ranging from 50-700 mg per day, more commonly 100-600 or 100-300 mg per day.

In one embodiment, compounds of the invention are administered in combination with a nitric oxide donor, examples of which include, but are not limited to nicorandil; organic nitrates such as pentaerythritol tetranitrate; and sydnonimines such as linsidomine and molsidomine.

In yet another embodiment, compounds of the invention are administered in combination with a non-steroidal anti-inflammatory agent (NSAID). Representative NSAIDs include, but are not limited to: acemetacin, acetyl salicylic acid, alclofenac, alminoprofen, amfenac, amiprilose, aloxiprin, anirolac, apazone, azapropazone, benorilate, benoxaprofen, bezpiperylon, broperamole, bucloxic acid, carprofen, clidanac, diclofenac, diflunisal, diftalone, enolicam, etodolac, etoricoxib, fenbufen, fenclofenac, fenclozic acid, fenoprofen, fentiazac, feprazone, flufenamic acid, flufenisal, fluprofen, flurbiprofen, furofenac, ibufenac, ibuprofen, indomethacin, indoprofen, isoxepac, isoxicam, ketoprofen, ketorolac, lofemizole, lornoxicam, meclofenamate, meclofenamic acid, mefenamic acid, meloxicam, mesalamine, miroprofen, mofebutazone, nabumetone, naproxen, niflumic acid, oxaprozin, oxpinac, oxyphenbutazone, phenylbutazone, piroxicam, pirprofen, pranoprofen, salsalate, sudoxicam, sulfasalazine, sulindac, suprofen, tenoxicam, tiopinac, tiaprofenic acid, tioxaprofen, tolfenamic acid, tolmetin, triflumidate, zidometacin, zomepirac, and combinations thereof. In a particular embodiment, the NSAID is selected from etodolac, flurbiprofen, ibuprofen, indomethacin, ketoprofen, ketorolac, meloxicam, naproxen, oxaprozin, piroxicam, and combinations thereof.

In one embodiment, compounds of the invention are administered in combination with an N-methyl d-aspartate (NMDA) receptor antagonist, examples of which include, by way of illustration and not limitation, including amantadine, dextromethorphan, dextropropoxyphene, ketamine, ketobemidone, memantine, methadone, and so forth.

In still another embodiment, compounds of the invention are administered in combination with an opioid receptor agonist (also referred to as opioid analgesics). Representative opioid receptor agonists include, but are not limited to: buprenorphine, butorphanol, codeine, dihydrocodeine, fentanyl, hydrocodone, hydromorphone, levallorphan, levorphanol, meperidine, methadone, morphine, nalbuphine, nalmefene, nalorphine, naloxone, naltrexone, nalorphine, oxycodone, oxymorphone, pentazocine, propoxyphene, tramadol, and combinations thereof. In certain embodiments, the opioid receptor agonist is selected from codeine, dihydrocodeine, hydrocodone, hydromorphone, morphine, oxycodone, oxymorphone, tramadol, and combinations thereof.

In a particular embodiment, compounds of the invention are administered in combination with a phosphodiesterase (PDE) inhibitor, particularly a PDE-V inhibitor. Representative PDE-V inhibitors include, but are not limited to, avanafil, lodenafil, mirodenafil, sildenafil (Revatio®), tadalafil (Adcirca®), vardenafil (Levitra®), and udenafil.

In another embodiment, compounds of the invention are administered in combination with a prostaglandin analog (also referred to as prostanoids or prostacyclin analogs). Representative prostaglandin analogs include, but are not limited to, beraprost sodium, bimatoprost, epoprostenol, iloprost, latanoprost, tafluprost, travoprost, and treprostinil, with bimatoprost, latanoprost, and tafluprost being of particular interest.

In yet another embodiment, compounds of the invention are administered in combination with a prostaglandin receptor agonist, examples of which include, but are not limited to, bimatoprost, latanoprost, travoprost, and so forth.

Compounds of the invention may also be administered in combination with a renin inhibitor, examples of which include, but are not limited to, aliskiren, enalkiren, remikiren, and combinations thereof.

In another embodiment, compounds of the invention are administered in combination with a selective serotonin reuptake inhibitor (SSRI). Representative SSRIs include, but are not limited to: citalopram and the citalopram metabolite desmethylcitalopram, dapoxetine, escitalopram (e.g., escitalopram oxalate), fluoxetine and the fluoxetine desmethyl metabolite norfluoxetine, fluvoxamine (e.g., fluvoxamine maleate), paroxetine, sertraline and the sertraline metabolite demethylsertraline, and combinations thereof.

In one embodiment, compounds of the invention are administered in combination with a 5-HT_(1D) serotonin receptor agonist, examples of which include, by way of illustration and not limitation, triptans such as almotriptan, avitriptan, eletriptan, frovatriptan, naratriptan, rizatriptan, sumatriptan, and zolmitriptan.

In one embodiment, compounds of the invention are administered in combination with a sodium channel blocker, examples of which include, by way of illustration and not limitation, carbamazepine, fosphenytoin, lamotrigine, lidocaine, mexiletine, oxcarbazepine, phenytoin, and combinations thereof.

In one embodiment, compounds of the invention are administered in combination with a soluble guanylate cyclase stimulator or activator, examples of which include, but are not limited to ataciguat, riociguat, and combinations thereof.

In one embodiment, compounds of the invention are administered in combination with a tricyclic antidepressant (TCA), examples of which include, by way of illustration and not limitation, amitriptyline, amitriptylinoxide, butriptyline, clomipramine, demexiptiline, desipramine, dibenzepin, dimetacrine, dosulepin, doxepin, imipramine, imipraminoxide, lofepramine, melitracen, metapramine, nitroxazepine, nortriptyline, noxiptiline, pipofezine, propizepine, protriptyline, quinupramine, and combinations thereof.

In one embodiment, compounds of the invention are administered in combination with a vasopressin receptor antagonist, examples of which include, by way of illustration and not limitation, conivaptan and tolvaptan.

Combined secondary therapeutic agents may also be helpful in further combination therapy with compounds of the invention. For example, compounds of the invention can be combined with a diuretic and an ARB, or a calcium channel blocker and an ARB, or a diuretic and an ACE inhibitor, or a calcium channel blocker and a statin. Specific examples include, a combination of the ACE inhibitor enalapril (in the maleate salt form) and the diuretic hydrochlorothiazide, which is sold under the mark Vaseretic®, or a combination of the calcium channel blocker amlodipine (in the besylate salt form) and the ARB olmesartan (in the medoxomil prodrug form), or a combination of a calcium channel blocker and a statin, all may also be used with the compounds of the invention. Other therapeutic agents such as α₂-adrenergic receptor agonists and vasopressin receptor antagonists may also be helpful in combination therapy. Exemplary α₂-adrenergic receptor agonists include clonidine, dexmedetomidine, and guanfacine.

The following formulations illustrate representative pharmaceutical compositions of the invention.

Exemplary Hard Gelatin Capsules for Oral Administration

A compound of the invention (50 g), 440 g spray-dried lactose and 10 g magnesium stearate are thoroughly blended. The resulting composition is then loaded into hard gelatin capsules (500 mg of composition per capsule). Alternately, a compound of the invention (20 mg) is thoroughly blended with starch (89 mg), microcrystalline cellulose (89 mg) and magnesium stearate (2 mg). The mixture is then passed through a No. 45 mesh U.S. sieve and loaded into a hard gelatin capsule (200 mg of composition per capsule).

Alternately, a compound of the invention (30 g), a secondary agent (20 g), 440 g spray-dried lactose and 10 g magnesium stearate are thoroughly blended, and processed as described above.

Exemplary Gelatin Capsule Formulation for Oral Administration

A compound of the invention (100 mg) is thoroughly blended with polyoxyethylene sorbitan monooleate (50 mg) and starch powder (250 mg). The mixture is then loaded into a gelatin capsule (400 mg of composition per capsule). Alternately, a compound of the invention (70 mg) and a secondary agent (30 mg) are thoroughly blended with polyoxyethylene sorbitan monooleate (50 mg) and starch powder (250 mg), and the resulting mixture loaded into a gelatin capsule (400 mg of composition per capsule).

Alternately, a compound of the invention (40 mg) is thoroughly blended with microcrystalline cellulose (Avicel PH 103; 259.2 mg) and magnesium stearate (0.8 mg). The mixture is then loaded into a gelatin capsule (Size #1, White, Opaque) (300 mg of composition per capsule).

Exemplary Tablet Formulation for Oral Administration

A compound of the invention (10 mg), starch (45 mg) and microcrystalline cellulose (35 mg) are passed through a No. 20 mesh U.S. sieve and mixed thoroughly. The granules so produced are dried at 50-60° C. and passed through a No. 16 mesh U.S. sieve. A solution of polyvinylpyrrolidone (4 mg as a 10% solution in sterile water) is mixed with sodium carboxymethyl starch (4.5 mg), magnesium stearate (0.5 mg), and talc (1 mg), and this mixture is then passed through a No. 16 mesh U.S. sieve. The sodium carboxymethyl starch, magnesium stearate and talc are then added to the granules. After mixing, the mixture is compressed on a tablet machine to afford a tablet weighing 100 mg.

Alternately, a compound of the invention (250 mg) is thoroughly blended with microcrystalline cellulose (400 mg), silicon dioxide fumed (10 mg), and stearic acid (5 mg). The mixture is then compressed to form tablets (665 mg of composition per tablet).

Alternately, a compound of the invention (400 mg) is thoroughly blended with cornstarch (50 mg), croscarmellose sodium (25 mg), lactose (120 mg), and magnesium stearate (5 mg). The mixture is then compressed to form a single-scored tablet (600 mg of composition per tablet).

Alternately, a compound of the invention (100 mg) is thoroughly blended with cornstarch (100 mg) with an aqueous solution of gelatin (20 mg). The mixture is dried and ground to a fine powder. Microcrystalline cellulose (50 mg) and magnesium stearate (5 mg) are then admixed with the gelatin formulation, granulated and the resulting mixture compressed to form tablets (100 mg of the compound of the invention per tablet).

Exemplary Suspension Formulation for Oral Administration

The following ingredients are mixed to form a suspension containing 100 mg of the compound of the invention per 10 mL of suspension:

Ingredients Amount Compound of the invention 1.0 g Fumaric acid 0.5 g Sodium chloride 2.0 g Methyl paraben 0.15 g Propyl paraben 0.05 g Granulated sugar 25.5 g Sorbitol (70% solution) 12.85 g Veegum ® K (magnesium aluminum silicate) 1.0 g Flavoring 0.035 mL Colorings 0.5 mg Distilled water q.s. to 100 mL

Exemplary Liquid Formulation for Oral Administration

A suitable liquid formulation is one with a carboxylic acid-based buffer such as citrate, lactate and maleate buffer solutions. For example, a compound of the invention (which may be pre-mixed with DMSO) is blended with a 100 mM ammonium citrate buffer and the pH adjusted to pH 5, or is blended with a 100 mM citric acid solution and the pH adjusted to pH 2. Such solutions may also include a solubilizing excipient such as a cyclodextrin, for example the solution may include 10 wt % hydroxypropyl-β-cyclodextrin.

Other suitable formulations include a 5% NaHCO₃ solution, with or without cyclodextrin.

Exemplary Injectable Formulation for Administration by Injection

A compound of the invention (0.2 g) is blended with 0.4 M sodium acetate buffer solution (2.0 mL). The pH of the resulting solution is adjusted to pH 4 using 0.5 N aqueous hydrochloric acid or 0.5 N aqueous sodium hydroxide, as necessary, and then sufficient water for injection is added to provide a total volume of 20 mL. The mixture is then filtered through a sterile filter (0.22 micron) to provide a sterile solution suitable for administration by injection.

Exemplary Compositions for Administration by Inhalation

A compound of the invention (0.2 mg) is micronized and then blended with lactose (25 mg). This blended mixture is then loaded into a gelatin inhalation cartridge. The contents of the cartridge are administered using a dry powder inhaler, for example.

Alternately, a micronized compound of the invention (10 g) is dispersed in a solution prepared by dissolving lecithin (0.2 g) in demineralized water (200 mL). The resulting suspension is spray dried and then micronized to form a micronized composition comprising particles having a mean diameter less than about 1.5 μm. The micronized composition is then loaded into metered-dose inhaler cartridges containing pressurized 1,1,1,2-tetrafluoroethane in an amount sufficient to provide about 10 μg to about 500 μg of the compound of the invention per dose when administered by the inhaler.

Alternately, a compound of the invention (25 mg) is dissolved in citrate buffered (pH 5) isotonic saline (125 mL). The mixture is stirred and sonicated until the compound is dissolved. The pH of the solution is checked and adjusted, if necessary, to pH 5 by slowly adding aqueous 1 N NaOH. The solution is administered using a nebulizer device that provides about 10 μg to about 500 μg of the compound of the invention per dose.

EXAMPLES

The following Preparations and Examples are provided to illustrate specific embodiments of the invention. These specific embodiments, however, are not intended to limit the scope of the invention in any way unless specifically indicated. The following abbreviations have the following meanings unless otherwise indicated and any other abbreviations used herein and not defined have their standard, generally accepted meaning:

-   -   AcOH acetic acid     -   Cbz carbobenzyloxy (—C(O)O-benzyl)     -   DCM dichloromethane or methylene chloride     -   DIPEA N,N-diisopropylethylamine     -   DMF N,N-dimethylformamide     -   Dnp 2,4-dinitrophenyl     -   EDCI N-(3-dimethylaminopropyl)-N″-ethylcarbodiimide     -   EtOAc ethyl acetate     -   EtOH ethanol     -   HATU N,N,N′,N′-tetramethyl-O-(7-azabenzotriazol-1-yl)uronium         hexafluorophosphate     -   HEPES 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid     -   HOBt 1-hydroxybenzotriazole hydrate     -   Mca (7-methoxycoumarin-4-yl)acyl     -   MeCN acetonitrile     -   MeOH methanol     -   Pd(PPh₃)₄ tetrakis(triphenylphosphine)palladium(0)     -   SilicaCat® DPP-Pd silica based diphenylphosphine palladium (II)         catalyst     -   TFA trifluoroacetic acid     -   THF tetrahydrofuran

Unless noted otherwise, all materials, such as reagents, starting materials and solvents, were purchased from commercial suppliers (such as Sigma-Aldrich, Fluka Riedel-de Haën, and the like) and were used without further purification.

Reactions were run under nitrogen atmosphere, unless noted otherwise. The progress of reactions were monitored by thin layer chromatography (TLC), analytical high performance liquid chromatography (anal. HPLC), and mass spectrometry, the details of which are given in specific examples. Solvents used in analytical HPLC were as follows: solvent A was 98% H₂O/2% MeCN/1.0 mL/L TFA; solvent B was 90% MeCN/10% H₂O/1.0 mL/L TFA.

Reactions were worked up as described specifically in each preparation for example; commonly reaction mixtures were purified by extraction and other purification methods such as temperature-, and solvent-dependent crystallization, and precipitation. In addition, reaction mixtures were routinely purified by preparative HPLC, typically using Microsorb C18 and Microsorb BDS column packings and conventional eluents. Progress of reactions was typically measured by liquid chromatography mass spectrometry (LCMS). Characterization of isomers were done by Nuclear Overhauser effect spectroscopy (NOE). Characterization of reaction products was routinely carried out by mass and ¹H-NMR spectrometry. For NMR measurement, samples were dissolved in deuterated solvent (CD₃OD, CDCl₃, or DMSO-d₆), and ¹H-NMR spectra were acquired with a Varian Gemini 2000 instrument (400 MHz) under standard observation conditions. Mass spectrometric identification of compounds was typically conducted using an electrospray ionization method (ESMS) with an Applied Biosystems (Foster City, Calif.) model API 150 EX instrument or an Agilent (Palo Alto, Calif.) model 1200 LC/MSD instrument.

Preparation 1 [(R)-1-(2-Chloro-benzyl)-2-cyano-2-hydroxy-ethyl]-carbamic Acid Benzyl Ester

To a suspension of (R)-2-amino-3-(2-chloro-phenyl)-propionic acid (100.0 g, 0.5 mol) in water (1 L) was added dropwise 4N aqueous NaOH (125 mL) at 0° C. Then a solution of N-(benzyloxycarbonyloxy)succinimide (125.0 g, 0.5 mol) in acetone (300 mL) was added in one portion. The pH of the mixture was maintained at 8˜9 by addition of 3N aqueous NaOH. After the mixture was stirred for 4 hours, the pH was adjusted to 1 with 6N HCl and the mixture was extracted with EtOAc (2×500 mL). The combined extracts were washed with 1N HCl (2×500 mL), dried over anhydrous Na₂SO₄ and concentrated to yield compound (1) as a white solid (155.0 g).

To a solution of compound (1) (80.0 g, 240 μmol) in DCM (500 mL) was added EDCI (50.6 g, 264 μmol), HOBt (35.6 g, 264 μmol), N,O-dimethylhydroxylamine hydrochloride (51.5 g, 528 μmol) and triethylamine (111 mL, 790 μmol). The mixture was stirred at room temperature overnight. Then the mixture was washed with 2N HCl (3×500 mL) and saturated aqueous NaHCO₃ (3×500 mL), respectively. The organic layer was dried over anhydrous Na₂SO₄ and concentrated to yield compound (2) as a yellow oil (71.0 g).

To a suspension of LiAlH₄ (7.2 g, 188 μmol) in THF (800 mL) was added dropwise a solution of compound (2) (71.0 g, 188 μmol) in THF (200 mL) at −20° C. The mixture was stirred at −20° C. for 2 hours. Then the reaction was quenched carefully with 1N HCl. The mixture was extracted with EtOAc (2×600 mL) and the combined extracts were dried over anhydrous Na₂SO₄ and concentrated to yield compound (3) (59.0 g), which was used directly without further purification.

To a solution of compound (3) (59.0 g, 188 μmol) in THF (500 mL) was added aqueous NaHSO₃ (19.5 g in 500 mL of water) and the mixture was stirred at 0° C. overnight. NaCN (9.2 g, 188 μmol) was added and the resulting mixture was stirred for 3 hours. The mixture was extracted with EtOAc (2×500 mL) and the combined extracts were dried over anhydrous Na₂SO₄ and concentrated to yield the title compound (64.0 g), which was used directly without further purification.

Preparation 2 (2R,3R)-3-Amino-4-(2-chloro-phenyl)-2-hydroxy-butyric Acid Methyl Ester

A mixture of [(R)-1-(2-chloro-benzyl)-2-cyano-2-hydroxy-ethyl]-carbamic acid benzyl ester (55.0 g, 157 μmol) in dioxane (300 mL) and 6N HCl (300 mL) was heated under reflux overnight. The solvent was removed under reduced pressure and the residue was dissolved in a 3N HCl-MeOH solution. The resulting mixture was refluxed for 4 hours and the solvent was removed under reduced pressure. The residue was taken up in EtOAc (500 mL) and aqueous NaHCO₃ (500 mL). The organic layer was separated, dried over anhydrous Na₂SO₄ and concentrated to give a mixture of the title compound and its (R,S)-isomer, which was subjected to flash column chromatography (DCM:MeOH=100:1 to 50:1) to yield the title compound (8.1 g).

¹H NMR (CDCl₃): δ 7.37 (m, 1H), 7.22 (m, 3H), 4.30 (d, J=3.3 Hz, 1H), 3.80 (s, 3H), 3.51 (m, 1H), 2.96 (m, 1H), 2.71 (m, 1H), 2.06 (br s, 2H). MS (m/z): 244 [M+H]⁺.

Preparation 3 (2R,3R)-3-Amino-4-(2-chloro-phenyl)-2-hydroxy-butyric Acid Ethyl Ester

A mixture of [(R)-1-(2-chloro-benzyl)-2-cyano-2-hydroxy-ethyl]-carbamic acid benzyl ester (64.0 g, 188 μmol) in dioxane (300 mL) and 6N HCl (300 mL) was heated under reflux overnight. The solvent was removed under reduced pressure and the residue was dissolved in a 3N HCl-EtOH solution. The resulting mixture was refluxed for 4 hours and the solvent was removed under reduced pressure. The residue was taken up in EtOAc (500 mL) and aqueous NaHCO₃ (500 mL). The organic layer was separated, dried over anhydrous Na₂SO₄ and concentrated to give a mixture of the title compound and its (R,S)-isomer, which was subjected to flash column chromatography (DCM:MeOH=100:1 to 50:1) to yield the title compound (9.7 g).

¹H NMR (CDCl₃): δ 7.37 (m, 1H), 7.22 (m, 3H), 4.25 (m, 3H), 3.50 (s, 1H), 2.97 (d, J=10.8 Hz, 1H), 2.74 (t, J=11.4 Hz, 1H), 2.06 (br s, 2H), 1.35 (t, J=7.1 Hz, 3H). MS (m/z): 258 [M+H]⁺.

Example 1

2-{2-[(1R,2R)-2-Carboxy-1-(2-chloro-benzyl)-2-hydroxy-ethylcarbamoyl]-pyridin-4-yl}-benzoic Acid (R¹=—OH; R⁴¹=H)

Methyl 4-bromopicolinate (167 mg, 772 μmol. 1.0 eq.) was mixed with 2-t-butoxycarbonylphenylboronic acid pinacol ester (282 mg, 927 μmol, 1.2 eq.) in toluene (1.1 mL). MeOH (409 μL) was added, followed by K₂CO₃ (214 mg, 1.5 mmol, 2.0 eq.) predissolved in water (202 μL). The mixture was stirred, and the reaction vessel was capped, placed under vacuum and purged with nitrogen. Pd(PPh₃)₄ (89.2 mg, 77.2 μmol) was added. The vessel was recapped and heated at 110° C. for 50 minutes. The organic layer was removed and the product was evaporated under vacuum. THF (3 mL) and 1 M aqueous NaOH (1.5 mL, 2.0 eq.) was added and the mixture was stirred for 2 hours. EtOAc was added and mixture was acidified with saturated NH₄Cl. The organic layers were extracted, dried and evaporated to yield 4-(2-t-butoxycarbonyl-phenyl)-pyridine-2-carboxylic acid.

4-(2-t-Butoxycarbonyl-phenyl)-pyridine-2-carboxylic acid (30 mg, 0.1 mmol, 1.0 eq.) and (2R,3R)-3-amino-4-(2-chloro-phenyl)-2-hydroxy-butyric acid ethyl ester (16.8 mg, 65.1 μmol, 1.0 eq.) were combined with DIPEA (52.4 μL, 3.0 eq.) and HATU (26.7 mg, 70.2 μmol, 0.7 eq.) and DCM (0.8 mL), and the resulting mixture was stirred for 45 minutes at room temperature. The reaction was quenched with saturated NH₄Cl, and the product was extracted with DCM, dried and evaporated to yield 2-{2-[(1R,2R)-1-(2-chloro-benzyl)-2-ethoxycarbonyl-2-hydroxy-ethylcarbamoyl]-pyridin-4-yl}-benzoic acid t-butyl ester.

2-{2-[(1R,2R)-1-(2-Chloro-benzyl)-2-ethoxycarbonyl-2-hydroxy-ethylcarbamoyl]-pyridin-4-yl}-benzoic acid t-butyl ester (104 mg, 192 μmol, 1.0 eq.) was combined with 1:1 TFA/DCM (1 mL each) and the resulting mixture was stirred for 1 hour. The solvent was evaporated and THF (3 mL) and 1 M aqueous NaOH (577 μL, 3.0 eq.) were added and the resulting mixture was stirred for 2 hours. The solvent was evaporated and AcOH (2 mL) was added. The product was then purified by preparative HPLC to yield the title compound as a TFA salt (2.4 mg, 96% purity). MS m/z [M+H]⁺ calc'd for C₂₃H₁₉ClN₂O₆, 455.09; found 455.

2-{2-[(1R,2R)-1-(2-Chloro-benzyl)-2-ethoxycarbonyl-2-hydroxy-ethylcarbamoyl]-pyridin-4-yl}-benzoic acid ethyl ester (R¹=—OCH₂CH₃; R⁴¹=—CH₂CH₃)

4-Bromopyridine-2-carboxylic acid (93.9 mg, 465 μmol, 1.0 eq.) and (2R,3R)-3-amino-4-(2-chloro-phenyl)-2-hydroxy-butyric acid ethyl ester (120 mg, 465 μmol, 1.0 eq.) were combined with DIPEA (434 μL) and HATU (177 mg, 465 μmol, 1.0 eq.) in DCM (4 mL) and stirred for 45 minutes at room temperature. The reaction was quenched with saturated NaHCO₃ and extracted with DCM, dried and evaporated to yield (2R,3R)-3-[(4-bromo-pyridine-2-carbonyl)-amino]-4-(2-chloro-phenyl)-2-hydroxy-butyric acid ethyl ester. This crude product was mixed with ethyl 2-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzoate (154 mg, 558 μmol, 1.2 eq.) and toluene (540 μL). EtOH (0.3 mL) was added, followed by K₂CO₃ (128 mg, 930 μmol, 2.0 eq.) predissolved in water (0.1 mL). The mixture was stirred, and the reaction vessel was capped, placed under vacuum and purged with nitrogen. Pd(PPh₃)₄ (53.7 mg, 46.5 μmol) was added. The vessel was recapped and heated at 100° C. for 30 minutes. The organic layer was retained and the product was evaporated under vacuum then purified using preparative HPLC to yield the title compound as a TFA salt (30 mg, 97% purity). MS m/z [M+H]⁺ calc'd for C₂₇H₂₇ClN₂O₆, 511.16; found 512.2.

2-{2-[(1R,2R)-1-(2-Chloro-benzyl)-2-ethoxycarbonyl-2-hydroxy-ethylcarbamoyl]-pyridin-4-yl}-benzoic acid (R¹=—OCH₂CH₃; R⁴¹=H)

Crude 2-{2-[(1R,2R)-1-(2-chloro-benzyl)-2-ethoxycarbonyl-2-hydroxy-ethylcarbamoyl]-pyridin-4-yl}-benzoic acid ethyl ester was dissolved in DCM/TFA (0.3 mL each) and stirred for 2 hours. The solvent was evaporated and the product was purified using preparative HPLC to yield the title compound as a TFA salt (75 mg, 97% purity). MS m/z [M+H]⁺ calc'd for C₂₅H₂₃ClN₂O₆, 483.12; found 483.

Example 2

Following the procedures described in the examples herein, and substituting the appropriate starting materials and reagents, compounds having formula Ic, were prepared as the parent compound or as a TFA salt:

MS m/z: [M + H]⁺ Ex. R¹ R⁴ R⁶ Formula calcd found 1 OH

CH₃ C₂₄H₂₁ClN₂O₆ 469.11 469.0

-   1.     2-{2-[(1R,2R)-2-Carboxy-1-(2-chloro-benzyl)-2-hydroxy-ethylcarbamoyl]-6-methyl-pyridin-4-yl}-benzoic     acid (TFA salt)

MS m/z: [M + H]⁺ Ex. R¹ R⁴ R⁶ Formula calcd found 2 OH

H C₂₃H₁₉ClN₂O₆ 455.09 456 3 OCH₂CH₃

H C₂₇H₂₇ClN₂O₆ 511.16 511 4 OH

CH₃ C₂₄H₂₁ClN₂O₆ 469.11 470

-   2.     2-{4-[(1R,2R)-2-Carboxy-1-(2-chloro-benzyl)-2-hydroxy-ethylcarbamoyl]-pyridin-2-yl}-benzoic     acid (TFA salt) -   3.     2-{4-[(1R,2R)-1-(2-Chloro-benzyl)-2-ethoxycarbonyl-2-hydroxy-ethylcarbamoyl]-pyridin-2-yl}-benzoic     acid ethyl ester (TFA salt) -   4.     2-{4-[(1R,2R)-2-Carboxy-1-(2-chloro-benzyl)-2-hydroxy-ethylcarbamoyl]-6-methyl-pyridin-2-yl}-benzoic     acid (TFA salt)

MS m/z: [M + H]⁺ Ex. R¹ R⁴ R⁶ Formula calcd found 5 OH

H C₂₃H₁₉ClN₂O₆ 455.09 455.2 6 OCH₂CH₃

H C₂₅H₂₃ClN₂O₆ 483.12 483.2 7 OH

OH C₂₃H₁₉ClN₂O₇ 471.09 471.0

-   5.     2-{5-[(1R,2R)-2-Carboxy-1-(2-chloro-benzyl)-2-hydroxy-ethylcarbamoyl]-pyridin-3-yl}-benzoic     acid (TFA salt) -   6.     2-{5-[(1R,2R)-1-(2-Chloro-benzyl)-2-ethoxycarbonyl-2-hydroxy-ethylcarbamoyl]-pyridin-3-yl}-benzoic     acid (TFA salt) -   7.     2-{5-[(1R,2R)-2-Carboxy-1-(2-chloro-benzyl)-2-hydroxy-ethylcarbamoyl]-2-hydroxy-pyridin-3-yl}-benzoic     acid (TFA salt)

MS m/z: [M + H]⁺ Ex. R¹ R⁴ R⁶ Formula calcd found  8 OH

H C₂₃H₁₉ClN₂O₆ 455.09 455    9 OH

Cl C₂₃H₁₈Cl₂N₂O₆ 489.05 490   10 OH

F C₂₃H₁₈ClFN₂O₆ 473.08 473.0

-   8.     2-{5-[(1R,2R)-2-Carboxy-1-(2-chloro-benzyl)-2-hydroxy-ethylcarbamoyl]-pyridin-2-yl}-benzoic     acid (TFA salt) -   9.     2-{5-[(1R,2R)-2-Carboxy-1-(2-chloro-benzyl)-2-hydroxy-ethylcarbamoyl]-3-chloro-pyridin-2-yl}-benzoic     acid (TFA salt) -   10.     2-{5-[(1R,2R)-2-Carboxy-1-(2-chloro-benzyl)-2-hydroxy-ethylcarbamoyl]-3-fluoro-pyridin-2-yl}-benzoic     acid (TFA salt)

MS m/z: [M + H]⁺ Ex. R¹ R⁴ Formula calcd found 11 OH OH C₁₄H₁₃ClN₂O₆ 341.05 341.0 12 OH

C₂₀H₁₆Cl₂N₂O₅ 435.04 436.0 13 OH

C₂₀H₁₆Cl₂N₂O₅ 435.04 435.4 14 OH

C₂₀H₁₆Cl₂N₂O₅ 435.04 435.2

-   11.     (2R,3R)-4-(2-Chloro-phenyl)-2-hydroxy-3-[(3-hydroxy-isoxazole-5-carbonyl)-amino]-butyric     acid (TFA salt) -   12.     (2R,3R)-4-(2-Chloro-phenyl)-3-{[3-(2-chloro-phenyl)-isoxazole-5-carbonyl]-amino}-2-hydroxy-butyric     acid (TFA salt) -   13.     (2R,3R)-4-(2-Chloro-phenyl)-3-{[3-(3-chloro-phenyl)-isoxazole-5-carbonyl]-amino}-2-hydroxy-butyric     acid (TFA salt) -   14.     (2R,3R)-4-(2-Chloro-phenyl)-3-{[3-(4-chloro-phenyl)-isoxazole-5-carbonyl]-amino}-2-hydroxy-butyric     acid (TFA salt)

MS m/z: [M + H]⁺ Ex. R¹ R⁴ Formula calcd found 15 OH

C₂₀H₁₆Cl₂N₂O₅ 435.04 435.2

-   15.     (2R,3R)-4-(2-Chloro-phenyl)-3-{[5-(2-chloro-phenyl)-isoxazole-3-carbonyl]-amino}-2-hydroxy-butyric     acid (TFA salt)

MS m/z: [M + H]⁺ Ex. R¹ R⁴ R⁵ Formula calcd found 16 OH ═O

C₂₁H₁₇Cl₂FN₂O₆ 483.04 483.2 17 OH ═O

C₂₂H₁₉ClF₂N₂O₇ 497.08 497.4

-   16.     (2R,3R)-3-{[2-(4-Chloro-2-fluoro-benzyl)-3-oxo-2,3-dihydro-isoxazole-5-carbonyl]-amino}-4-(2-chloro-phenyl)-2-hydroxy-butyric     acid (TFA salt) -   17.     (2R,3R)-4-(2-Chloro-phenyl)-3-{[2-(2,3-difluoro-4-methoxy-benzyl)-3-oxo-2,3-dihydro-isoxazole-5-carbonyl]-amino}-2-hydroxy-butyric     acid (TFA salt)

MS m/z: [M + H]⁺ Ex. R¹ R⁴ Formula calcd found 18 OH

C₂₀H₁₇ClN₂O₅ 401.08 401.2

-   18.     (2R,3R)-4-(2-Chloro-phenyl)-2-hydroxy-3-[(2-phenyl-oxazole-5-carbonyl)-amino]-butyric     acid (TFA salt)

MS m/z: [M + H]⁺ Ex. R¹ R⁵ Formula calcd found 19 OH H C₁₃H₁₃ClN₄O₄ 325.06 325.0

-   19.     (2S,3R)-4-(2-Chloro-phenyl)-2-hydroxy-3-[(3H-[1,2,3]triazole-4-carbonyl)-amino]-butyric     acid (TFA salt)

MS m/z: [M + H]⁺ Ex. R¹ R⁵ Formula calcd found 20 OH

C₂₀H₁₉ClN₄O₄ 415.11 415.2 21 OH OH C₁₃H₁₃ClN₄O₅ 341.06 341.0

-   20.     (2R,3R)-3-[(1-Benzyl-1H-[1,2,3]triazole-4-carbonyl)-amino]-4-(2-chloro-phenyl)-2-hydroxy-butyric     acid (TFA salt) -   21.     (2R,3R)-4-(2-Chloro-phenyl)-2-hydroxy-3-[(1-hydroxy-1H-[1,2,3]triazole-4-carbonyl)-amino]-butyric     acid (TFA salt)

MS m/z: [M + H]⁺ Ex. R¹ R⁴ R⁵ Formula calcd found 22 OH CH₃

C₂₀H₁₈Cl₂N₄O₄ 449.07 450.0

-   22.     (2R,3R)-4-(2-Chloro-phenyl)-3-{[1-(4-chloro-phenyl)-5-methyl-1H-[1,2,4]triazole-3-carbonyl]-amino}-2-hydroxy-butyric     acid (TFA salt)

MS m/z: [M + H]⁺ Ex. R¹ R⁴ R⁵ Formula calcd found 23 OH ═O

C₂₀H₁₇Cl₂FN₄O₅ 483.06 483.4

-   23.     (2R,3R)-3-{[1-(4-Chloro-2-fluoro-benzyl)-5-oxo-4,5-dihydro-1H-[1,2,4]triazole-3-carbonyl]-amino}-4-(2-chloro-phenyl)-2-hydroxy-butyric     acid (TFA salt)

MS m/z: [M + H]⁺ Ex. R¹ Formula calcd found 24 OH C₂₁H₁₈ClN₃O₅ 428.09 428.3 25 OCH₂CH₃ C₂₃H₂₂ClN₃O₅ 456.12 456.2

-   24.     (2R,3R)-4-(2-Chloro-phenyl)-2-hydroxy-3-[(5-oxa-3,3a-diaza-cyclopenta[a]naphthalene-2-carbonyl)-amino]-butyric     acid (TFA salt) -   25.     (2R,3R)-4-(2-Chloro-phenyl)-2-hydroxy-3-[(5-oxa-3,3a-diaza-cyclopenta[a]naphthalene-2-carbonyl)-amino]-butyric     acid ethyl ester (TFA salt)

MS m/z: [M + H]⁺ Ex. R¹ Formula calcd found 26 OH C₂₂H₂₀ClN₃O₅ 442.11 442.4

-   26.     (2R,3R)-4-(2-Chloro-phenyl)-3-[(4,5-dihydro-6-oxa-3,3a-diaza-benzo[e]azulene-2-carbonyl)-amino]-2-hydroxy-butyric     acid (TFA salt)

MS m/z: [M + H]⁺ Ex. R¹ R⁴ Formula calcd found 27 OH F C₁₇H₁₄ClFN₄O₄ 393.07 393.0 28 OH Cl C₁₇H₁₄Cl₂N₄O₄ 409.04 410.0 29 OH H C₁₇H₁₅ClN₄O₄ 375.08 375.0 30 OH CH₃ C₁₈H₁₇ClN₄O₄ 389.09 389.0

-   27.     (2S,3R)-4-(2-Chloro-phenyl)-3-[(7-fluoro-3H-benzotriazole-5-carbonyl)-amino]-2-hydroxy-butyric     acid (TFA salt) -   28.     (2S,3R)-3-[(7-Chloro-3H-benzotriazole-5-carbonyl)-amino]-4-(2-chloro-phenyl)-2-hydroxy-butyric     acid (TFA salt) -   29.     (2S,3R)-3-[(3H-Benzotriazole-5-carbonyl)-amino]-4-(2-chloro-phenyl)-2-hydroxy-butyric     acid (TFA salt) -   30.     (2S,3R)-4-(2-Chloro-phenyl)-2-hydroxy-3-[(7-methyl-3H-benzotriazole-5-carbonyl)-amino]-butyric     acid (TFA salt)

MS m/z: [M + H]⁺ Ex. R¹ R⁴ Formula calcd found 31 OH COOH C₁₈H₁₅ClN₄O₆ 419.07 419.2

-   31.     6-[(1R,2R)-2-Carboxy-1-(2-chloro-benzyl)-2-hydroxy-ethylcarbamoyl]-3H-benzotriazole-4-carboxylic     acid (TFA salt)

MS m/z: [M + H]⁺ Ex. R¹ R⁴ Formula calcd found 32 OH COOH C₁₆H₁₄ClNO₇ 368.05 368.0

-   32.     5-[(1R,2R)-2-Carboxy-1-(2-chloro-benzyl)-2-hydroxy-ethylcarbamoyl]-furan-2-carboxylic     acid (TFA salt)

MS m/z: [M + H]⁺ Ex. R¹ R⁴ Formula calcd found 33 OH Br C₁₈H₁₅BrClN₃O₄ 451.99 452.0

-   33.     (2R,3R)-3-[(3-Bromo-imidazo[1,2-a]pyridine-6-carbonyl)-amino]-4-(2-chloro-phenyl)-2-hydroxy-butyric     acid (TFA salt)

MS m/z: [M + H]⁺ Ex. R¹ R⁴ Formula calcd found 34 OH OH C₁₅H₁₄ClN₃O₅ 352.06 352.2

-   34.     (2R,3R)-4-(2-Chloro-phenyl)-2-hydroxy-3-[5-hydroxy-pyrazine-2-carbonyl)-amino]-butyric     acid (TFA salt)

MS m/z: [M + H]⁺ Ex. R¹ R⁴ Formula calcd found 35 OH OH C₁₅H₁₄ClN₃O₅ 352.06 352.4

-   35.     (2R,3R)-4-(2-Chloro-phenyl)-2-hydroxy-3-[2-hydroxy-pyrimidine-5-carbonyl)-amino]-butyric     acid (TFA salt)

MS m/z: [M + H]⁺ Ex. R¹ Formula calcd found 36 OH C₁₇H₁₅ClN₄O₄ 375.08 375.0

-   36.     (2R,3R)-4-(2-Chloro-phenyl)-2-hydroxy-3-[(3H-imidazo[4,5-b]pyridine-6-carbonyl)-amino]-butyric     acid (TFA salt)

MS m/z: [M + H]⁺ Ex. R¹ Formula calcd found 37 OH C₁₆H₁₄ClN₅O₄ 376.07 n.d. 38 OCH₂CH₃ C₁₈H₁₈ClN₅O₄ 404.10 404.2 n.d. = not determined

-   37.     (2R,3R)-4-(2-Chloro-phenyl)-2-hydroxy-3-[(1H-[1,2,3]triazolo[4,5-b]pyridine-6-carbonyl)-amino]-butyric     acid (TFA salt) -   38.     (2R,3R)-4-(2-Chloro-phenyl)-2-hydroxy-3-[(1H-[1,2,3]triazolo[4,5-b]pyridine-6-carbonyl)-amino]-butyric     acid ethyl ester (TFA salt)

MS m/z: [M + H]⁺ Ex. R¹ R⁴ Formula calcd found 39 OH ═O C₁₅H₁₆ClN₃O₅ 354.08 354.0

-   39.     (2R,3R)-4-(2-Chloro-phenyl)-2-hydroxy-3-[(6-oxo-1,4,5,6-tetrahydro-pyridazine-3-carbonyl)-amino]-butyric     acid

Preparation 4 (2R,3R)-3-Amino-2-hydroxy-4-(2-trifluoromethyl-phenyl)-butyric Acid Ethyl Ester

To a suspension of (R)-2-amino-3-(2-trifluoromethyl-phenyl)-propionic acid (25.0 g, 107 μmol) in water (230 mL) was added dropwise 4N aqueous NaOH (25 mL) at 0° C. Then a solution of N-(benzyloxycarbonyloxy)succinimide (26.8 g, 107 μmol) in acetone (100 mL) was added in one portion. The pH of the mixture was maintained at 8˜9 by addition of 3N aqueous NaOH. After the mixture was stirred for 4 hours, the pH was adjusted to 1 with 6N HCl and the mixture was extracted with EtOAc (2×200 mL). The combined extracts were washed with 1N HCl (2×200 mL), dried over anhydrous Na₂SO₄ and concentrated to yield compound (1) as a white solid (35.0 g).

To a solution of compound (1) (35.0 g, 95 μmol) in DCM (300 mL) was added EDCI (20.0 g, 104 μmol), HOBt (14.0 g, 104 μmol), N,O-dimethylhydroxylamine hydrochloride (20.0 g, 208 μmol) and triethylamine (33.0 g, 312 μmol). The mixture was stirred at room temperature overnight. Then the mixture was washed with 2N HCl (3×250 mL) and saturated aqueous NaHCO₃ (3×200 mL), respectively. The organic layer was dried over anhydrous Na₂SO₄ and concentrated to yield compound (2) (35.0 g).

To a suspension of LiAlH₄ (3.3 g, 85 μmol) in THF (300 mL) was added dropwise a solution of compound (2) (35.0 g, 85 μmol) in THF (150 mL) at −20° C. The mixture was stirred at −20° C. for 2.5 hours. Then the reaction was quenched carefully with 1N HCl. The mixture was extracted with EtOAc (2×300 mL) and the combined extracts were dried over anhydrous Na₂SO₄ and concentrated to yield compound (3) (29.8 g), which was used directly without further purification.

To a solution of compound (3) (29.8 g, 85 μmol) in THF (250 mL) was added aqueous NaHSO₃ (8.84 g in 250 mL of water) and the mixture was stirred at 0° C. overnight. NaCN (4.2 g, 85 μmol) was added and the resulting mixture was stirred for 3 hours. The mixture was extracted with EtOAc (2×300 mL) and the combined extracts were dried over anhydrous Na₂SO₄ and concentrated to yield compound (4) (32.1 g, quantitative), which was used directly without further purification.

A mixture of compound (4) (32.1 g, 85 μmol) in dioxane (200 mL) and 6N HCl (200 mL) was heated under reflux overnight. The solvent was removed under reduced pressure and the residue was dissolved in a 3N HCl-EtOH solution. The resulting mixture was refluxed overnight and the solvent was removed under reduced pressure. The residue was taken up in EtOAc (300 mL) and aqueous NaHCO₃ (300 mL). The organic layer was separated, dried over anhydrous Na₂SO₄ and concentrated to give a mixture of the title compound and its (R,S)-isomer, which was subjected to flash column chromatography (DCM:MeOH=100:1 to 50:1) to yield the title compound (6.0 g).

¹H NMR (CDCl₃): δ 7.68 (d, J=7.8 Hz, 1H), 7.52 (t, J=7.5 Hz, 1H), 7.37 (m, 2H), 4.31 (m, 3H), 3.42 (m, 1H), 3.00 (dd, J=2.4, 14.4 Hz, 1H), 2.79 (m, 1H), 1.37 (t, J=7.1 Hz, 3H). MS (m/z): 292 [M+H]⁺.

Preparation 5 1H-[1,2,3]-Triazolo[4,5-b]pyridine-6-carboxylic Acid

Methyl 6-amino-5-nitronicotinate (5.0 g, 25.4 mmol, 1.0 eq.) was combined with acetic anhydride (100 g) and sulfuric acid (1 mL). The resulting solution was stirred for 2 hours at room temperature. The solids were collected by filtration and washed with 10% NaHCO₃ (aq.). The solid was dried in an oven under reduced pressure to yield compound (1) as a yellow solid (3.9 g).

Compound (1) (3.9 g, 16.3 μmol, 1.0 eq.) was dissolved in MeOH (200 mL), and palladium carbon (0.5 g) was added. The resulting solution was stirred overnight at room temperature, under hydrogen. The solids were filtered out and the resulting mixture was concentrated under vacuum to yield compound (2) as a white solid (3.0 g).

Compound (2) (3.0 g, 14.4 mmol, 1.0 equiv) was dissolved in sulfuric acid/H₂O (1:4) (100 mL). A solution of NaNO₂ (1.1 g, 15.7 mmol, 1.1 eq.) in water (15 mL) was added dropwise with stirring at −5˜0 CC over 5 minutes. The resulting solution was stirred for 1 hour at 0° C. in a water/ice bath. The solids were collected by filtration and washed with water. The solid was dried in an oven under reduced pressure to yield compound (3) as a yellow solid (2.5 g).

Compound (3) (3.5 g, 19.7 mmol, 1.0 eq.) was dissolved in THF (20 mL). A solution of NaOH (5.6 g, 140.0 mmol, 7.1 eq.) in water (50 mL) was added, and the resulting solution was stirred for 4 hours at room temperature. The resulting mixture was concentrated under vacuum, then washed with EtOAc (2×30 mL). The pH value of the solution was adjusted to 3 with HCl (5 mol/L). The solids were collected by filtration, and dried in an oven under reduced pressure to yield the title compound as a white solid (2.7 g). ES, m/z: 165 [M+H]⁺; ¹HNMR (DMSO, 300 Hz, ppm) 13.70 (s, 1H), 9.19 (s, 1H), 8.92 (s, 1H).

Example 3 (2R,3R)-2-Hydroxy-3-[(3H-[1,2,3]-triazolo[4,5-b]pyridine-6-carbonyl)-amino]-4-(2-trifluoromethyl-phenyl)-butyric acid

1H-[1,2,3]Triazolo[4,5-b]pyridine-6-carboxylic acid (86 mg, 520 μmmol, 1.0 eq.) was combined with HATU (239 mg, 629 μmol, 1.2 eq.) in DMF (3 mL) and stirred at room temperature for 15 minutes. (2R,3R)-3-Amino-2-hydroxy-4-(2-trifluoromethyl-phenyl)-butyric acid ethyl ester (153 mg, 524 μmol, 1.0 eq.) in DIPEA (182 μL, 2.0 eq.) and DMF (3 mL) was added and the resulting mixture was stirred overnight at room temperature. 2 M aqueous NaOH (3 mL) and THF (3 mL) were added and the mixture was heated at 40° C. overnight. The product was purified by preparative HPLC to yield the title compound as a TFA salt (68.8 mg, 100% purity). MS m/z [M+H]⁺ calc'd for C₁₇H₁₄F₃N₅O₄, 410.10; found 410.2.

Example 4

2-[6-((1R,2R)-1-Benzyl-2-carboxy-2-hydroxy-ethylcarbamoyl)-pyridazin-3-yl]-benzoic Acid (R¹=—OH; R⁴¹=H)

6-Chloropyridazine-3-carboxylic acid (78.9 mg, 497 μmol, 1.0 eq.), DIPEA (273 μL, 3.2 eq.), HATU (189 mg, 497 μmol, 1.0 eq.), and (2R,3R)-3-amino-2-hydroxy-4-phenyl-butyric acid ethyl ester (129 mg, 497 μmol, 1.0 eq.) were combined in DCM (2 mL) and stirred for 1 hour. The crude reaction was chromatographed using a gradient (0-80% EtOAc/Hex) to obtain (2R,3R)-3-[(6-chloro-pyridazine-3-carbonyl)-amino]-2-hydroxy-4-phenyl-butyric acid ethyl ester.

(2R,3R)-3-[(6-Chloro-pyridazine-3-carbonyl)-amino]-2-hydroxy-4-phenyl-butyric acid ethyl ester (172 mg, 474 μmol. 1.0 eq.) was mixed with 2-t-butoxycarbonylphenyl-boronic acid pinacol ester (173 mg, 568 μmol, 1.2 eq.) in toluene (661 μL). EtOH (361 μL) was added, followed by K₂CO₃ (131 mg, 947 μmol, 2.0 eq.) predissolved in water (124 μL). The mixture was stirred, and the reaction vessel was capped, placed under vacuum and purged with nitrogen. Pd(PPh₃)₄ (54.7 mg, 47.4 μmol) was added. The vessel was recapped and heated at 100° C. for 30 minutes. The organic layer was removed and the product was evaporated under vacuum to yield 2-[6-((1R,2R)-1-benzyl-2-ethoxycarbonyl-2-hydroxy-ethylcarbamoyl)-pyridazin-3-yl]-benzoic acid t-butyl ester.

2-[6-((1R,2R)-1-benzyl-2-ethoxycarbonyl-2-hydroxy-ethylcarbamoyl)-pyridazin-3-yl]-benzoic acid t-butyl ester (30 mg, 59.3 μmol) was combined with TFA and DCM 1:1 (0.5 mL each) and the resulting mixture was stirred for 30 minutes. The solvent was evaporated and the product was dissolved in THF (0.5 mL). 5 M NaOH (0.3 mL) was added and the mixture was stirred overnight. AcOH was added to achieve a pH of 5 (˜1 mL). The product was then purified by preparative HPLC to yield the title compound (1 mg, 98% purity). MS m/z [M+H]⁺ calc'd for C₂₂H₁₉N₃O₆, 422.13; found 422.

2-[6-((1R,2R)-1-Benzyl-2-ethoxycarbonyl-2-hydroxy-ethylcarbamoyl)-pyridazin-3-yl]-benzoic Acid (R¹=—OCH₂CH₃; R⁴¹=H)

2-[6-((1R,2R)-1-benzyl-2-ethoxycarbonyl-2-hydroxy-ethylcarbamoyl)-pyridazin-3-yl]-benzoic acid t-butyl ester (97.3 mg, 192 μmol) was combined with TFA and DCM 1:1 (1 mL each) and the resulting mixture was stirred for 1 hour. The solvent was evaporated and THF (3 mL) and 1 M aqueous NaOH (577 μL, 3.0 eq.) were added. The mixture was stirred for 2 hours and EtOAc was added. The aqueous layer was extracted out and the organic was washed with NaOH (0.2 mL), stirred and the aqueous layer extracted and added to the first fraction. The aqueous was acidified to pH ˜5 with concentrated HCl, forming a gummy solid. The aqueous was removed and the solid was dissolved in AcOH and purified by preparative HPLC to yield the title compound (15 mg, 97% purity). MS m/z [M+H]⁺ calc'd for C₂₄H₂₃N₃O₆, 450.16; found 450.2.

2-[6-((1R,2R)-1-Benzyl-2-ethoxycarbonyl-2-hydroxy-ethylcarbamoyl)-pyridazin-3-yl]-benzoic Acid Ethyl Ester (R¹=—OCH₂CH₃; R⁴¹=—CH₇CH₃)

6-Chloropyridazine-3-carboxylic acid (71 mg, 440 μmol, 1.0 eq.), K₂CO₃ (185 mg, 1.3 μmol, 3.0 eq.), and 2-(4,4,5,5-tetramethyl-[1,3,2]dioxaborolan-2-yl)-benzoic acid ethyl ester (148 mg, 535 μmol, 1.2 eq.) were combined with EtOH (1 mL) and water (0.3 mL). The mixture was stirred, and the reaction vessel was capped, placed under vacuum and purged with nitrogen. SilicaCat® DPP-Pd (280 μmol/g loading; 286 mg, 80.2 μmol) was added. The vessel was recapped and microwaved at 100° C. for 20 minutes. The solvent was removed and the product filtered. The pH was adjusted to ˜4 with 1N HCl. HATU (136 mg, 356 μmol, 0.8 eq.), DIPEA (233 mL, 3.0 eq.), and (2R,3R)-3-amino-2-hydroxy-4-phenyl-butyric acid ethyl ester (99.5 mg, 446 μmol, 1.0 eq.) were combined in DCM (2 mL) and stirred for 2 hours. AcOH was added and the product was purified by preparative HPLC to yield the title compound as a TFA salt (1.8 mg, 95% purity). MS m/z [M+H]⁺ calc'd for C₂₆H₂₇N₃O₆, 478.19; found 478.

2-[6-((1R,2R)-1-Benzyl-2-butoxycarbonyl-2-hydroxy-ethylcarbamoyl)-pyridazin-3-yl]-benzoic Acid (R¹=—O(CH₂)₃CH₃; R⁴¹=H)

(2R,3R)-3-t-Butoxycarbonylamino-2-hydroxy-4-phenyl-butyric acid (70.9 mg, 240 μmol, 1.0 eq.) and 1-butanol (2 mL, 20 mmol) were combined in 4 M HCl in 1,4-dioxane (0.4 mL, 2 mmol), and stirred for 2 hours at 60° C. The solvent was evaporated and the reaction mixture was azeotroped with toluene. 6-Chloropyridazine-3-carboxylic acid (38.1 mg, 240 μmol, 1.0 eq.), DIPEA (134 μL, 3.2 eq.), and HATU (91.3 mg, 240 μmol, 1.0 eq.) were combined in DCM (5 mL) and stirred for 5 minutes at room temperature. This mixture was then added to the azeotroped mixture and stirred for 1 hour. The reaction was quenched with saturated NH₄Cl. The product was extracted with DCM, dried and evaporated, then purified by chromatography (0-60% EtOAc/hexanes gradient) to yield (2R,3R)-3-[((6-chloro-pyridazine-3-carbonyl)-amino]-2-hydroxy-4-phenyl-butyric acid butyl ester.

(2R,3R)-3-[(6-Chloro-pyridazine-3-carbonyl)-amino]-2-hydroxy-4-phenyl-butyric acid butyl ester (90 mg, 230 μmol, 1.0 eq.) was mixed with 2-t-butoxycarbonylphenyl-boronic acid pinacol ester (83.8 mg, 276 μmol, 1.2 eq.) in toluene (320 μL). 1-Butanol (274 μL) was added, followed by K₂CO₃ (63.5 mg, 459 μmol) predissolved in water (60 μL). The mixture was stirred, and the reaction vessel was capped, placed under vacuum and purged with nitrogen. Pd(PPh₃)₄ (26.5 mg, 23 μmol) was added. The vessel was recapped and microwaved at 100° C. for 40 minutes. The organic layer was removed, AcOH was added, and the product was purified by preparative HPLC to yield the title compound as a TFA salt (41 mg, 95% purity). MS m/z [M+H]⁺ calc'd for C₂₆H₂₇N₃O₆, 478.19; found 478.2.

Example 5

Following the procedures described in the examples herein, and substituting the appropriate starting materials and reagents, compounds having formula Ie, were prepared as the parent compound or as a TFA salt:

MS m/z: [M + H]⁺ Ex. R¹ R⁴ R⁵ Formula calcd found 1 OH ═O H C₂₂H₂₃N₃O₅ 410.16 410.2

-   1.     (2R,3R)-2-Hydroxy-3-[(4-oxo-1,2,3,3a,4,5-hexahydro-pyrrolo[1,2-a]quinoxaline-8-carbonyl)-amino]-4-phenyl-butyric     acid

MS m/z: [M + H]⁺ Ex. R¹ R⁴ R⁵ R⁶ Formula calcd found 2 OH (CH₂)₂—CH₃

CH₃ C₂₈H₂₉BrN₄O₄ 565.14 565.6 3 OH (CH₂)₂—CH₃

CH₃ C₂₈H₃₀N₄O₄ 487.23 487.6 4 OCH₂—CH₃ (CH₂)₂—CH₃

CH₃ C₃₀H₃₄N₄O₄ 515.26 515.4 5 OH (CH₂)₂—CH₃ CH₂COOH CH₃ C₂₄H₂₇N₃O₆ 454.19 454.2 6 OH (CH₂)₂—CH₃

CH₃ C₃₀H₃₃N₃O₅ 516.24 516.2 7 OH (CH₂)₂—CH₃

CH₃ C₃₀H₃₃N₃O₅ 516.24 516.2 8 OH (CH₂)₂—CH₃ CH₂—CONH₂ CH₃ C₂₄H₂₈N₄O₅ 453.21 453.2 9 OH CH₂—CH₃ H Cl C₂₀H₂₀ClN₃O₄ 402.11 402.0

-   2.     (2R,3R)-3-{[3-(6-Bromo-pyridin-3-ylmethyl)-7-methyl-2-propyl-3H-benzoimidazole-5-carbonyl]-amino}-2-hydroxy-4-phenyl-butyric     acid (TFA salt) -   3.     (2R,3R)-2-Hydroxy-3-[(7-methyl-2-propyl-3-pyridin-3-ylmethyl-3H-benzoimidazole-5-carbonyl)-amino]-4-phenyl-butyric     acid (TFA salt) -   4.     (2R,3R)-2-Hydroxy-3-[(7-methyl-2-propyl-3-pyridin-3-ylmethyl-3H-benzoimidazole-5-carbonyl)-amino]-4-phenyl-butyric     acid ethyl ester (TFA salt) -   5.     (2R,3R)-3-[(3-Carboxymethyl-7-methyl-2-propyl-3H-benzoimidazole-5-carbonyl)-amino]-2-hydroxy-4-phenyl-butyric     acid (TFA salt) -   6.     (2R,3R)-2-Hydroxy-3-{[3-(2-methoxy-benzyl)-7-methyl-2-propyl-3H-benzoimidazole-5-carbonyl]-amino}-4-phenyl-butyric     acid (TFA salt) -   7.     (2R,3R)-2-Hydroxy-3-{[3-(4-methoxy-benzyl)-7-methyl-2-propyl-3H-benzoimidazole-5-carbonyl]-amino}-4-phenyl-butyric     acid (TFA salt) -   8.     (2R,3R)-3-[(3-Carbamoylmethyl-7-methyl-2-propyl-3H-benzoimidazole-5-carbonyl)-amino]-2-hydroxy-4-phenyl-butyric     acid (TFA salt) -   9.     (2R,3R)-3-[(7-Chloro-2-ethyl-3H-benzoimidazole-5-carbonyl)-amino]-2-hydroxy-4-phenyl-butyric     acid

MS m/z: [M + H]⁺ Ex. R¹ R⁴ R⁵ R⁶ Formula calcd found 10 OCH₂CH₃ CH₂CH₃ H Cl C₂₂H₂₄ClN₃O₄ 430.15 430.0

-   10.     (2R,3R)-3-[(7-Chloro-2-ethyl-1H-benzoimidazole-5-carbonyl)-amino]-2-hydroxy-4-phenyl-butyric     acid ethyl ester

MS m/z: [M + H]⁺ Ex. R¹ R⁴ R⁵ Formula calcd found 11 OH H H C₁₇H₁₆N₄O₄ 341.12 341.2 12 OCH₂CH₃ H H C₁₉H₂₀N₄O₄ 369.15 369.2 13 OH Cl H C₁₇H₁₅ClN₄O₄ 375.08 375.0 14 OCH₂CH₃ Cl H C₁₉H₁₉ClN₄O₄ 403.11 403.0 15 OH CH₃ H C₁₈H₁₈N₄O₄ 355.13 355.2

-   11.     (2R,3R)-3-[(3H-Benzotriazole-5-carbonyl)-amino]-2-hydroxy-4-phenyl-butyric     acid (TFA salt) -   12.     (2R,3R)-3-[(3H-Benzotriazole-5-carbonyl)-amino]-2-hydroxy-4-phenyl-butyric     acid ethyl ester (TFA salt) -   13.     (2R,3R)-3-[(7-Chloro-3H-benzotriazole-5-carbonyl)-amino]-2-hydroxy-4-phenyl-butyric     acid -   14.     (2R,3R)-3-[(7-Chloro-3H-benzotriazole-5-carbonyl)-amino]-2-hydroxy-4-phenyl-butyric     acid ethyl ester -   15.     (2R,3R)-2-Hydroxy-3-[(7-methyl-3H-benzotriazole-5-carbonyl)-amino]-4-phenyl-butyric     acid (TFA salt)

MS m/z: [M + H]⁺ Ex. R¹ R⁴ R⁵ Formula calcd found 16 OH F H C₁₇H₁₅FN₄O₄ 359.11 359.0

-   16.     (2R,3R)-3-[(7-Fluoro-1H-benzotriazole-5-carbonyl)-amino]-2-hydroxy-4-phenyl-butyric     acid (TFA salt)

MS m/z: [M + H]⁺ Ex. R¹ R⁴ R⁵ R⁶ Formula calcd found 17 OH (CH₂)₃CH₃ (CH₂)₃OH Cl C₂₁H₂₈ClN₃O₅ 438.17 438.2

-   17.     (2S,3R)-3-{[2-Butyl-5-chloro-3-(3-hydroxy-propyl)-3H-imidazole-4-carbonyl]-amino}-2-hydroxy-4-phenyl-butyric     acid (TFA salt)

MS m/z: [M + H]⁺ Ex. R¹ R⁴ Formula calcd found 18 OH COOH C₁₆H₁₅N₃O₆ 346.10 346.0 19 OH

C₂₂H₁₉N₃O₆ 422.13 422.0

-   18.     5-((1R,2R)-1-Benzyl-2-carboxy-2-hydroxy-ethylcarbamoyl)-pyrazine-2-carboxylic     acid (TFA salt) -   19.     2-[5-((1R,2R)-1-Benzyl-2-carboxy-2-hydroxy-ethylcarbamoyl)-pyrazin-2-yl]-benzoic     acid (TFA salt)

MS m/z: [M + H]⁺ Ex. R¹ R⁴ Formula calcd found 20 OH COOH C₁₇H₁₆N₂O₆ 345.10 345.0 21 OH

C₂₃H₂₀N₂O₆ 421.13 421.0

-   20.     5-((1R,2R)-1-Benzyl-2-carboxy-2-hydroxy-ethylcarbamoyl)-pyridine-2-carboxylic     acid (TFA salt) -   21.     2-[5-((1R,2R)-1-Benzyl-2-carboxy-2-hydroxy-ethylcarbamoyl)-pyridin-2-yl]-benzoic     acid (TFA salt)

MS m/z: [M + H]⁺ Ex. R¹ R⁴ Formula calcd found 22 OH

C₂₃H₂₀N₂O₆ 421.13 421.0 23 OCH₂CH₃

C₂₇H₂₈N₂O₆ 477.19 478.0

-   22.     2-[4-((1R,2R)-1-Benzyl-2-carboxy-2-hydroxy-ethylcarbamoyl)-pyridin-2-yl]-benzoic     acid (TFA salt)

23. 2-[4-((1R,2R)-1-Benzyl-2-ethoxycarbonyl-2-hydroxy-ethylcarbamoyl)-pyridin-2-yl]-benzoic acid ethyl ester (TFA salt)

MS m/z: [M + H]⁺ Ex. R¹ R⁴ Formula calcd found 24 OH

C₂₃H₂₀N₂O₆ 421.13 421

-   24.     2-[6-((1R,2R)-1-Benzyl-2-carboxy-2-hydroxy-ethylcarbamoyl)-pyridin-3-yl]-benzoic     acid (TFA salt)

MS m/z: [M + H]⁺ Ex. R¹ R⁴ Formula calcd found 25 OH

C₂₃H₂₀N₂O₆ 421.13 421

-   25.     2-[5-((1R,2R)-1-Benzyl-2-carboxy-2-hydroxy-ethylcarbamoyl)-pyridin-3-yl]-benzoic     acid (TFA salt)

MS m/z: [M + H]⁺ Ex. R¹ R⁴ Formula calcd found 26 OH

C₂₃H₂₀N₂O₆ 421.13 421.0 27 OCH₂CH₃

C₂₅H₂₄N₂O₆ 449.16 449.0 28 OCH₂CH₃

C₂₇H₂₈N₂O₆ 477.19 477.2

-   26.     2-[2-((1R,2R)-1-Benzyl-2-carboxy-2-hydroxy-ethylcarbamoyl)-pyridin-4-yl]-benzoic     acid (TFA salt) -   27.     2-[2-((1R,2R)-1-Benzyl-2-ethoxycarbonyl-2-hydroxy-ethylcarbamoyl)-pyridin-4-yl]-benzoic     acid (TFA salt) -   28.     2-[2-((1R,2R)-1-Benzyl-2-ethoxycarbonyl-2-hydroxy-ethylcarbamoyl)-pyridin-4-yl]-benzoic     acid ethyl ester (TFA salt)

MS m/z: [M + H]⁺ Ex. R¹ R⁵ Formula calcd found 29 OH H C₁₆H₁₅N₅O₄ 342.11 342.2

-   29.     (2R,3R)-2-Hydroxy-4-phenyl-3-[(1H-[1,2,3]triazolo[4,5-b]pyridine-6-carbonyl)-amino]-butyric     acid (TFA salt)

MS m/z: [M + H]⁺ Ex. R¹ Formula calcd found 30 OH C₁₈H₁₇N₃O₄ 340.12 340.0

-   30.     (2R,3R)-2-Hydroxy-4-phenyl-3-[(pyrrolo[1,2-c]pyrimidine-3-carbonyl)-amino]-butyric     acid (TFA salt)

MS m/z: [M + H]⁺ Ex. R¹ R⁴ Formula calcd found 31 OH COOH C₁₆H₁₅NO₆S 350.06 350.0

-   31.     5-((1R,2R)-1-Benzyl-2-carboxy-2-hydroxy-ethylcarbamoyl)-thiophene-2-carboxylic     acid

Preparation 6 (R)-3-Amino-4-(2-chloro-phenyl)-butyric Acid Ethyl Ester

(R)-3-Amino-4-(2-chloro-phenyl)-butyric acid (10 g, 50 mmol) was dissolved in absolute EtOH (250 mL). Concentrated HCl (2 mL) was added and the mixture was heated to reflux overnight. The product was vacuumed to dryness and azeotroped with toluene (4×50 mL), then placed under high vacuum overnight to yield the title compound as a white solid (10 g).

Example 6 2-{2-[(R)-1-Carboxymethyl-2-(2-chloro-phenyl)-ethylcarbamoyl]-pyridin-4-yl}-benzoic Acid

4-Bromopyridine-2-carboxylic acid (159 mg, 788 μmol, 1.0 eq.), DIPEA (439 μL, 3.2 eq.) and HATU (300 mg, 788 μmol, 1.0 eq.) were combined in DCM (20 mL) and stirred for 5 minutes at room temperature. (R)-3-amino-4-(2-chloro-phenyl)-butyric acid ethyl ester (200 mg, 827 mmol, 1.1 eq.) was added and the resulting mixture was stirred for 1 hour. The reaction was quenched with saturated NH₄Cl, then extracted with DCM, dried and evaporated to yield the crude intermediate. K₂CO₃ (163 mg, 1.2 mmol), 2-t-butoxycarbonylphenylboronic acid pinacol ester (264 mg, 867 μmol, 1.1 eq.), EtOH (1 mL), and water (0.3 mL) were added to the crude intermediate. The mixture was stirred, and the reaction vessel was capped, placed under vacuum and purged with nitrogen. SilicaCat® DPP-Pd (280 μmol/g loading; 563 mg, 158 μmol) was added. The vessel was recapped and microwaved at 100° C. for 30 minutes. The solvent was removed and the product filtered. 1:1 DCM:TFA was added and the resulting mixture was stirred at 40° C. for 2 hours. The solvent was evaporated and the crude product was dissolved in AcOH (1.5 mL) and purified by preparative HPLC to yield the title compound as a TFA salt (50.0 mg). MS m/z [M+H]⁺ calc'd for C₂₃H₁₉ClN₂O₅, 439.10. found 440.

Example 7

Following the procedures described in the examples herein, and substituting the appropriate starting materials and reagents, compounds having formula Ih, were prepared as TFA salts:

MS m/z: [M + H]⁺ Ex. R¹ R⁵ Formula calcd found 1 OH H C₁₃H₁₃ClN₄O₃ 309.07 309.0

-   1.     (R)-4-(2-Chloro-phenyl)-3-[(3H-[1,2,3]triazole-4-carbonyl)-amino]-butyric     acid

MS m/z: [M + H]⁺ Ex. R¹ R⁴ Formula calcd found 2 OH CH₃ C₁₈H₁₇ClN₄O₃ 373.10 373.0 3 OH H C₁₇H₁₅ClN₄O₃ 359.08 359.0 4 OH Cl C₁₇H₁₄Cl₂N₄O₃ 393.04 393.0

-   2.     (R)-4-(2-Chloro-phenyl)-3-[(7-methyl-3H-benzotriazole-5-carbonyl)-amino]-butyric     acid -   3.     (R)-3-[(3H-Benzotriazole-5-carbonyl)-amino]-4-(2-chloro-phenyl)-butyric     acid -   4.     (R)-3-[(7-Chloro-3H-benzotriazole-5-carbonyl)-amino]-4-(2-chloro-phenyl)-butyric     acid

MS m/z: [M + H]⁺ Ex. R¹ R⁴ Formula calcd found 5 OCH₂CH₃ H C₁₉H₁₉ClN₄O₃ 387.11 n.d. 6 OH F C₁₇H₁₄ClFN₄O₃ 377.07 377.0 n.d. = not determined

-   5.     (R)-3-[(1H-Benzotriazole-5-carbonyl)-amino]-4-(2-chloro-phenyl)-butyric     acid ethyl ester -   6.     (R)-4-(2-Chloro-phenyl)-3-[(7-fluoro-1H-benzotriazole-5-carbonyl)-amino]-butyric     acid

MS m/z: [M + H]⁺ Ex. R¹ Formula calcd found 7 OH C₁₆H₁₄ClN₅O₃ 360.08 360.0

-   7.     (R)-4-(2-Chloro-phenyl)-3-[(1H-[1,2,3]triazolo[4,5-b]pyridine-6-carbonyl)-amino]-butyric     acid

MS m/z: [M + H]⁺ Ex. R¹ R⁴ Formula calcd found 8 OH OH C₁₅H₁₄ClN₃O₄ 336.07 336.4

-   8.     (R)-4-(2-Chloro-phenyl)-3-[(2-hydroxy-pyrimidine-5-carbonyl)-amino]-butyric     acid

MS m/z: [M + H]⁺ Ex. R¹ R⁴ Formula calcd found 9 OH

C₂₂H₁₈ClN₃O₅ 440.09 440.0

-   9.     2-{6-[(R)-2-Carboxy-1-(2-chloro-benzyl)-ethylcarbamoyl]-pyridazin-3-yl}-benzoic     acid

Assay 1 In Vitro Assays for the Quantitation of Inhibitor Potencies at Human and Rat NEP, and Human ACE

The inhibitory activities of compounds at human and rat neprilysin (EC 3.4.24.11; NEP) and human angiotensin converting enzyme (ACE) were determined using in vitro assays as described below.

Extraction of NEP Activity from Rat Kidneys

Rat NEP was prepared from the kidneys of adult Sprague Dawley rats. Whole kidneys were washed in cold phosphate buffered saline (PBS) and brought up in ice-cold lysis buffer (1% Triton X-114, 150 mM NaCl, 50 mM tris(hydroxymethyl)aminomethane (Tris) pH 7.5; Bordier (1981) J. Biol. Chem. 256:1604-1607) in a ratio of 5 mL of buffer for every gram of kidney. Samples were homogenized on ice using a polytron hand held tissue grinder. Homogenates were centrifuged at 1000×g in a swinging bucket rotor for 5 minutes at 3° C. The pellet was resuspended in 20 mL of ice cold lysis buffer and incubated on ice for 30 minutes. Samples (15-20 mL) were then layered onto 25 mL of ice-cold cushion buffer (6% w/v sucrose, 50 mM pH 7.5 Tris, 150 mM NaCl, 0.06%, Triton X-114), heated to 37° C. for 3-5 minutes and centrifuged at 1000×g in a swinging bucket rotor at room temperature for 3 minutes. The two upper layers were aspirated off, leaving a viscous oily precipitate containing the enriched membrane fraction. Glycerol was added to a concentration of 50% and samples were stored at −20° C. Protein concentrations were quantitated using a BCA detection system with bovine serum albumin (BSA) as a standard.

Enzyme Inhibition Assays

Recombinant human NEP and recombinant human ACE were obtained commercially (R&D Systems, Minneapolis, Minn., catalog numbers 1182-ZN and 929-ZN, respectively). The fluorogenic peptide substrate Mca-D-Arg-Arg-Leu-Dap-(Dnp)-OH (Medeiros et al. (1997) Braz. J. Med. Biol. Res. 30:1157-62; Anaspec, San Jose, Calif.) and Abz-Phe-Arg-Lys(Dnp)-Pro-OH (Araujo et al. (2000) Biochemistry 39:8519-8525; Bachem, Torrance, Calif.) were used in the NEP and ACE assays respectively.

The assays were performed in 384-well white opaque plates at 37° C. using the fluorogenic peptide substrates at a concentration of 10 μM in Assay Buffer (NEP: 50 mM HEPES, pH 7.5, 100 mM NaCl, 0.01% polyethylene glycol sorbitan monolaurate (Tween-20), 10 μM ZnSO₄; ACE: 50 mM HEPES, pH 7.5, 100 mM NaCl, 0.01% Tween-20, 1 μM ZnSO₄). The respective enzymes were used at concentrations that resulted in quantitative proteolysis of 1 μM of substrate after 20 minutes at 37° C.

Test compounds were assayed over the range of concentrations from 10 μM to 20 pM. Test compounds were added to the enzymes and incubated for 30 minute at 37° C. prior to initiating the reaction by the addition of substrate. Reactions were terminated after 20 minutes of incubation at 37° C. by the addition of glacial acetic acid to a final concentration of 3.6% (v/v).

Plates were read on a fluorometer with excitation and emission wavelengths set to 320 nm and 405 nm, respectively. Inhibition constants were obtained by nonlinear regression of the data using the equation (GraphPad Software, Inc., San Diego, Calif.): v=v ₀/[1+(I/K′)] where v is the reaction rate, v_(o) is the uninhibited reaction rate, I is the inhibitor concentration and K′ is the apparent inhibition constant.

Compounds of the invention were tested in this assay and found to have pK_(i) values at human NEP as follows. In general, either the prodrug compounds did not inhibit the enzyme in this in vitro assay, or the prodrugs were not tested (n.d.) since activity would not be expected.

Ex. pK_(i) 1; R¹ = —OH; ≧9.0 R⁴¹ = H 1; R¹ = —OCH₂CH₃; n.d. R⁴¹ = —CH₂CH₃ 1; R¹ = —OCH₂CH₃; n.d. R⁴¹ = H 2-1 ≧9.0 2-2 ≧9.0 2-3 n.d. 2-4 8.0-8.9 2-5 8.0-8.9 2-6 n.d. 2-7 ≧9.0 2-8 8.0-8.9 2-9 8.0-8.9 2-10 8.0-8.9 2-11 8.0-8.9 2-12 8.0-8.9 2-13 8.0-8.9 2-14 8.0-8.9 2-15 8.0-8.9 2-16 8.0-8.9 2-17 8.0-8.9 2-18 7.0-7.9 2-19 7.0-7.9 2-20 7.0-7.9 2-21 7.0-7.9 2-22 7.0-7.9 2-23 7.0-7.9 2-24 8.0-8.9 2-25 n.d. 2-26 8.0-8.9 2-27 8.0-8.9 2-28 8.0-8.9 2-29 7.0-7.9 2-30 7.0-7.9 2-31 7.0-7.9 2-32 7.0-7.9 2-33 7.0-7.9 2-34 7.0-7.9 2-35 ≧9.0 2-36 7.0-7.9 2-37 8.0-8.9 2-38 n.d. 2-39 7.0-7.9 3 8.0-8.9 4; R¹ = —OH; 8.0-8.9 R⁴¹ = H 4; R¹ = —OCH₂CH₃; n.d. R⁴¹ = H 4; R¹ = —OCH₂CH₃; n.d. R⁴¹ = —CH₂CH₃ 4; R¹ = —O(CH₂)₃CH₃; n.d. R⁴¹ = H 5-1 7.0-7.9 5-2 7.0-7.9 5-3 7.0-7.9 5-4 n.d. 5-5 7.0-7.9 5-6 7.0-7.9 5-7 7.0-7.9 5-8 7.0-7.9 5-9 7.0-7.9 5-10 n.d. 5-11 7.0-7.9 5-12 n.d. 5-13 7.0-7.9 5-14 n.d. 5-15 7.0-7.9 5-16 7.0-7.9 5-17 n.d. 5-18 7.0-7.9 5-19 7.0-7.9 5-20 7.0-7.9 5-21 8.0-8.9 5-22 8.0-8.9 5-23 n.d. 5-24 7.0-7.9 5-25 8.0-8.9 5-26 8.0-8.9 5-27 n.d. 5-28 n.d. 5-29 7.0-7.9 5-30 7.0-7.9 5-31 8.0-8.9 6 8.0-8.9 7-1 7.0-7.9 7-2 7.0-7.9 7-3 7.0-7.9 7-4 7.0-7.9 7-5 n.d. 7-6 7.0-7.9 7-7 7.0-7.9 7-8 8.0-8.9 7-9 7.0-7.9

Assay 2 Pharmacodynamic (PD) Assay for ACE and NEP Activity in Anesthetized Rats

Male, Sprague Dawley, normotensive rats are anesthetized with 120 mg/kg (i.p.) of inactin. Once anesthetized, the jugular vein, carotid artery (PE 50 tubing) and bladder (flared PE 50 tubing) catheters are cannulated and a tracheotomy is performed (Teflon Needle, size 14 gauge) to faciliate spontaneous respiration. The animals are then allowed a 60 minute stablization period and kept continuously infused with 5 mL/kg/h of saline (0.9%) throughout, to keep them hydrated and ensure urine production. Body temperature is maintained throughout the experiment by use of a heating pad. At the end of the 60 minute stabilization period, the animals are dosed intravenously (i.v.) with two doses of AngI (1.0 μg/kg, for ACE inhibitor activity) at 15 minutes apart. At 15 minutes post-second dose of AngI, the animals are treated with vehicle or test compound. Five minutes later, the animals are additionally treated with a bolus i.v. injection of atrial natriuretic peptide (ANP; 30 μg/kg). Urine collection (into pre-weighted eppendorf tubes) is started immediately after the ANP treatment and continued for 60 minutes. At 30 and 60 minutes into urine collection, the animals are re-challenged with AngI. Blood pressure measurements are done using the Notocord system (Kalamazoo, Mich.). Urine samples are frozen at −20° C. until used for the cGMP assay. Urine cGMP concentrations are determined by Enzyme Immuno Assay using a commercial kit (Assay Designs, Ann Arbor, Mich., Cat. No. 901-013). Urine volume is determined gravimetrically. Urinary cGMP output is calculated as the product of urine output and urine cGMP concentration. ACE inhibition is assessed by quantifying the % inhibition of pressor response to AngI. NEP inhibition is assessed by quantifying the potentiation of ANP-induced elevation in urinary cGMP output.

Assay 3 In Vivo Evaluation of Antihypertensive Effects in the Conscious SHR Model of Hypertension

Spontaneously hypertensive rats (SHR, 14-20 weeks of age) are allowed a minimum of 48 hours acclimation upon arrival at the testing site with free access to food and water. For blood pressure recording, these animals are surgically implanted with small rodent radiotransmitters (telemetry unit; DSI Models TA11PA-C40 or C50-PXT, Data Science Inc., USA). The tip of the catheter connected to the transmitter is inserted into the descending aorta above the iliac bifurcation and secured in place with tissue adhesive. The transmitter is kept intraperitoneally and secured to the abdominal wall while closing of the abdominal incision with a non-absorbable suture. The outer skin is closed with suture and staples. The animals are allowed to recover with appropriate post operative care. On the day of the experiment, the animals in their cages are placed on top of the telemetry receiver units to acclimate to the testing environment and baseline recording. After at least of 2 hours baseline measurement is taken, the animals are then dosed with vehicle or test compound and followed out to 24 hours post-dose blood pressure measurement. Data is recorded continuously for the duration of the study using Notocord software (Kalamazoo, Mich.) and stored as electronic digital signals. Parameters measured are blood pressure (systolic, diastolic and mean arterial pressure) and heart rate.

Assay 4 In Vivo Evaluation of Antihypertensive Effects in the Conscious DOCA-Salt Rat Model of Hypertension

CD rats (male, adult, 200-300 grams, Charles River Laboratory, USA) are allowed a minimum of 48 hours acclimation upon arrival at the testing site before they are placed on a high salt diet. One week after the start of the high salt diet (8% in food or 1% NaCl in drinking water), a deoxycorticosterone acetate (DOCA) pellet (100 mg, 90 days release time, Innovative Research of America, Sarasota, Fla.) is implanted subcutaneously and unilateral nephrectomy is performed. At this time, the animals are also surgically implanted with small rodent radiotransmitters for blood pressure measurement (see Assay 3 for details). The animals are allowed to recover with appropriate post operative care. Study design, data recording, and parameters measured is similar to that described for Assay 3.

Assay 5 In Vivo Evaluation of Antihypertensive Effects in the Conscious Dahl/SS Rat Model of Hypertension

Male, Dahl salt sensitive rats (Dahl/SS, 6-7 weeks of age from Charles River Laboratory, USA) are allowed at least 48 hours of acclimation upon arrival at the testing site before they were placed on a 8% NaCl high salt diet (TD.92012, Harlan, USA) then surgically implanted with small rodent radiotransmitters for blood pressure measurement (see Assay 3 for details). The animals are allowed to recover with appropriate post operative care. At approximately 4 to 5 weeks from the start of high salt diet, these animals are expected to become hypertensive. Once the hypertension level is confirmed, these animals are used for the study while continued with the high salt diet to maintain their hypertension level. Study design, data recording, and parameters measured is similar to that described in Assay 3.

While the present invention has been described with reference to specific aspects or embodiments thereof, it will be understood by those of ordinary skilled in the art that various changes can be made or equivalents can be substituted without departing from the true spirit and scope of the invention. Additionally, to the extent permitted by applicable patent statutes and regulations, all publications, patents and patent applications cited herein are hereby incorporated by reference in their entirety to the same extent as if each document had been individually incorporated by reference herein. 

The invention claimed is:
 1. A compound of formula I:

where: R² is OH; R³ is —CF₃; R¹ is —OR¹⁰; R¹⁰ is H or —C₁₋₆alkyl; X is pyridyltriazole; R⁴ is H; R⁵ is H; and R⁶ is H.
 2. A pharmaceutical composition comprising the compound of claim 1 and a pharmaceutically acceptable carrier.
 3. The pharmaceutical composition of claim 2, further comprising a therapeutic agent selected from adenosine receptor antagonists, α-adrenergic receptor antagonists, β₁-adrenergic receptor antagonists, β₂-adrenergic receptor agonists, dual-acting β-adrenergic receptor antagonist/α₁-receptor antagonists, advanced glycation end product breakers, aldosterone antagonists, aldosterone synthase inhibitors, aminopeptidase N inhibitors, androgens, angiotensin-converting enzyme inhibitors and dual-acting angiotensin-converting enzyme/neprilysin inhibitors, angiotensin-converting enzyme 2 activators and stimulators, angiotensin-II vaccines, anticoagulants, anti-diabetic agents, antidiarrheal agents, anti-glaucoma agents, anti-lipid agents, antinociceptive agents, anti-thrombotic agents, AT₁ receptor antagonists and dual-acting AT₁ receptor antagonist/neprilysin inhibitors and multifunctional angiotensin receptor blockers, bradykinin receptor antagonists, calcium channel blockers, chymase inhibitors, digoxin, diuretics, dopamine agonists, endothelin converting enzyme inhibitors, endothelin receptor antagonists, HMG-CoA reductase inhibitors, estrogens, estrogen receptor agonists and/or antagonists, monoamine reuptake inhibitors, muscle relaxants, natriuretic peptides and their analogs, natriuretic peptide clearance receptor antagonists, neprilysin inhibitors, nitric oxide donors, non-steroidal anti-inflammatory agents, N-methyl d-aspartate receptor antagonists, opioid receptor agonists, phosphodiesterase inhibitors, prostaglandin analogs, prostaglandin receptor agonists, renin inhibitors, selective serotonin reuptake inhibitors, sodium channel blocker, soluble guanylate cyclase stimulators and activators, tricyclic antidepressants, vasopressin receptor antagonists, and combinations thereof.
 4. The pharmaceutical composition of claim 3, wherein the therapeutic agent is an AT₁ receptor antagonist.
 5. A process for preparing the compound of claim 1, comprising the step of: (a) coupling compound 1 with compound 2:

to produce a compound of formula I or a pharmaceutically acceptable salt thereof.
 6. An intermediate useful in the synthesis of the compound of claim 1, having formula II:

where P is selected from —O—P¹; P¹ is a carboxy-protecting group selected from methyl, ethyl, t-butyl, benzyl, p-methoxybenzyl, 9-fluorenylmethyl, trimethylsilyl, t-butyldimethylsilyl, and diphenylmethyl; or a salt thereof.
 7. A method for treating hypertension, heart failure, or renal disease, comprising administering to a patient a therapeutically effective amount of the compound of claim
 1. 8. (2R,3R)-4-(2-chloro-phenyl)-2-hydroxy-3-[(3-hydroxy-isoxazole-5-carbonyl)-amino]-butyric acid.
 9. (2S,3R)-3-[(7-chloro-3H-benzotriazole-5-carbonyl)-amino]-4-(2-chloro-phenyl)-2-hydroxy-butyric acid.
 10. (R)-4-(2-chloro-phenyl)-3-[(2-hydroxy-pyrimidine-5-carbonyl)-amino]-butyric acid. 